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1.
Nat Commun ; 11(1): 6091, 2020 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-33257699

RESUMO

Adenocarcinoma at the gastroesophageal junction (ACGEJ) has dismal clinical outcomes, and there are currently few specific effective therapies because of limited knowledge on its genomic and transcriptomic alterations. The present study investigates genomic and transcriptomic changes in ACGEJ from Chinese patients and analyzes their drug vulnerabilities and associations with the survival time. Here we show that the major genomic changes of Chinese ACGEJ patients are chromosome instability promoted tumorigenic focal copy-number variations and COSMIC Signature 17-featured single nucleotide variations. We provide a comprehensive profile of genetic changes that are potentially vulnerable to existing therapeutic agents and identify Signature 17-correlated IFN-α response pathway as a prognostic marker that might have practical value for clinical prognosis of ACGEJ. These findings further our understanding on the molecular biology of ACGEJ and may help develop more effective therapeutic strategies.


Assuntos
Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Junção Esofagogástrica/metabolismo , Genômica , Transcriptoma , Adenocarcinoma/tratamento farmacológico , Idoso , Grupo com Ancestrais do Continente Asiático/genética , Instabilidade Cromossômica , Variações do Número de Cópias de DNA , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Feminino , Genes Neoplásicos/genética , Humanos , Interferon-alfa/metabolismo , Estimativa de Kaplan-Meier , Mutação , Prognóstico , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo
2.
Cell Rep ; 33(7): 108407, 2020 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-33207208

RESUMO

Individuals with Down syndrome (DS; trisomy 21) display hyperactivation of interferon (IFN) signaling and chronic inflammation, which could potentially be explained by the extra copy of four IFN receptor (IFNR) genes encoded on chromosome 21. However, the clinical effects of IFN hyperactivity in DS remain undefined. Here, we report that a commonly used mouse model of DS overexpresses IFNR genes and shows hypersensitivity to IFN ligands in diverse immune cell types. When treated repeatedly with a TLR3 agonist to induce chronic inflammation, these animals overexpress key IFN-stimulated genes, induce cytokine production, exhibit liver pathology, and undergo rapid weight loss. Importantly, the lethal immune hypersensitivity and cytokine production and the ensuing pathology are ameliorated by JAK1 inhibition. These results indicate that individuals with DS may experience harmful hyperinflammation upon IFN-inducing immune stimuli, as observed during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, pointing to JAK1 inhibition as a strategy to restore immune homeostasis in DS.


Assuntos
Azetidinas/uso terapêutico , Síndrome de Down/imunologia , Hipersensibilidade/tratamento farmacológico , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 2/antagonistas & inibidores , Inibidores de Proteínas Quinases/uso terapêutico , Sulfonamidas/uso terapêutico , Animais , Síndrome de Down/complicações , Feminino , Hipersensibilidade/etiologia , Hipersensibilidade/imunologia , Imunidade Inata , Interferon-alfa/metabolismo , Fígado/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores Toll-Like/metabolismo
3.
J Virol ; 94(23)2020 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-32938761

RESUMO

SARS-CoV-2, a novel coronavirus (CoV) that causes COVID-19, has recently emerged causing an ongoing outbreak of viral pneumonia around the world. While distinct from SARS-CoV, both group 2B CoVs share similar genome organization, origins to bat CoVs, and an arsenal of immune antagonists. In this report, we evaluate type I interferon (IFN-I) sensitivity of SARS-CoV-2 relative to the original SARS-CoV. Our results indicate that while SARS-CoV-2 maintains similar viral replication to SARS-CoV, the novel CoV is much more sensitive to IFN-I. In Vero E6 and in Calu3 cells, SARS-CoV-2 is substantially attenuated in the context of IFN-I pretreatment, whereas SARS-CoV is not. In line with these findings, SARS-CoV-2 fails to counteract phosphorylation of STAT1 and expression of ISG proteins, while SARS-CoV is able to suppress both. Comparing SARS-CoV-2 and influenza A virus in human airway epithelial cultures, we observe the absence of IFN-I stimulation by SARS-CoV-2 alone but detect the failure to counteract STAT1 phosphorylation upon IFN-I pretreatment, resulting in near ablation of SARS-CoV-2 infection. Next, we evaluated IFN-I treatment postinfection and found that SARS-CoV-2 was sensitive even after establishing infection. Finally, we examined homology between SARS-CoV and SARS-CoV-2 in viral proteins shown to be interferon antagonists. The absence of an equivalent open reading frame 3b (ORF3b) and genetic differences versus ORF6 suggest that the two key IFN-I antagonists may not maintain equivalent function in SARS-CoV-2. Together, the results identify key differences in susceptibility to IFN-I responses between SARS-CoV and SARS-CoV-2 that may help inform disease progression, treatment options, and animal model development.IMPORTANCE With the ongoing outbreak of COVID-19, differences between SARS-CoV-2 and the original SARS-CoV could be leveraged to inform disease progression and eventual treatment options. In addition, these findings could have key implications for animal model development as well as further research into how SARS-CoV-2 modulates the type I IFN response early during infection.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Interferon Tipo I/farmacologia , Interferon-alfa/farmacologia , Vírus da SARS/efeitos dos fármacos , Animais , Antivirais/antagonistas & inibidores , Antivirais/metabolismo , Betacoronavirus/imunologia , Betacoronavirus/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Humanos , Interferon Tipo I/antagonistas & inibidores , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Interferon-alfa/antagonistas & inibidores , Interferon-alfa/imunologia , Interferon-alfa/metabolismo , Fosforilação , Proteínas Recombinantes/farmacologia , Vírus da SARS/imunologia , Vírus da SARS/fisiologia , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , Células Vero , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral/efeitos dos fármacos
4.
Sci Rep ; 10(1): 12767, 2020 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-32728070

RESUMO

Recombinant interferon-α (IFN-α) treatment functionally cures chronic hepatitis B virus (HBV) infection in some individuals and suppresses virus replication in hepatocytes infected in vitro. We studied the antiviral effect of conditioned media (CM) from peripheral blood mononuclear cells (PBMCs) stimulated with agonists of Toll-like receptors (TLRs) 2, 7, 8 and 9. We found that CM from PBMCs stimulated with dual-acting TLR7/8 (R848) and TLR2/7 (CL413) agonists were more potent drivers of inhibition of HBe and HBs antigen secretion from HBV-infected primary human hepatocytes (PHH) than CM from PBMCs stimulated with single-acting TLR7 (CL264) or TLR9 (CpG-B) agonists. Inhibition of HBV in PHH did not correlate with the quantity of PBMC-produced IFN-α, but it was a complex function of multiple secreted cytokines. More importantly, we found that the CM that efficiently inhibited HBV production in freshly isolated PHH via various cytokine repertoires and mechanisms did not reduce covalently closed circular (ccc)DNA levels. We confirmed our data with a cell culture model based on HepG2-NTCP cells and the plasmacytoid dendritic cell line GEN2.2. Collectively, our data show the importance of dual-acting TLR agonists inducing broad cytokine repertoires. The development of poly-specific TLR agonists provides novel opportunities towards functional HBV cure.


Assuntos
Hepatite B Crônica/virologia , Hepatócitos/virologia , Leucócitos Mononucleares/metabolismo , Receptores Toll-Like/agonistas , Replicação Viral/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Citocinas/metabolismo , DNA Circular/metabolismo , Sistemas de Liberação de Medicamentos , Células Hep G2 , Vírus da Hepatite B/fisiologia , Humanos , Imunidade Inata/efeitos dos fármacos , Interferon-alfa/metabolismo , Receptores Toll-Like/metabolismo
5.
Science ; 369(6504): 718-724, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32661059

RESUMO

Coronavirus disease 2019 (COVID-19) is characterized by distinct patterns of disease progression that suggest diverse host immune responses. We performed an integrated immune analysis on a cohort of 50 COVID-19 patients with various disease severity. A distinct phenotype was observed in severe and critical patients, consisting of a highly impaired interferon (IFN) type I response (characterized by no IFN-ß and low IFN-α production and activity), which was associated with a persistent blood viral load and an exacerbated inflammatory response. Inflammation was partially driven by the transcriptional factor nuclear factor-κB and characterized by increased tumor necrosis factor-α and interleukin-6 production and signaling. These data suggest that type I IFN deficiency in the blood could be a hallmark of severe COVID-19 and provide a rationale for combined therapeutic approaches.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Interferon alfa-2/metabolismo , Interferon-alfa/metabolismo , Interferon beta/metabolismo , Pneumonia Viral/imunologia , Adulto , Idoso , Betacoronavirus/fisiologia , Infecções por Coronavirus/virologia , Estado Terminal , Estudos Transversais , Feminino , Perfilação da Expressão Gênica , Humanos , Imunidade Inata , Inflamação , Interleucina-6/metabolismo , Masculino , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Pandemias , Pneumonia Viral/virologia , Transdução de Sinais , Subpopulações de Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Carga Viral
6.
PLoS One ; 15(7): e0235488, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32667932

RESUMO

Mycobacterium tuberculosis (M.tb) infection stimulates the release of cytokines, including interferons (IFNs). IFNs are initiators, regulators, and effectors of innate and adaptive immunity. Accordingly, the expression levels of Type I (α, ß) and II (γ) IFNs, among untreated tuberculosis (TB) patients and household contacts (HHC) clinically free of TB was assessed. A total of 264 individuals (TB patients-123; HHC-86; laboratory volunteers-55; Treated TB patients-36) were enrolled for this study. IFN-α mRNA expression levels predominated compared to IFN-γ and IFN-ß among untreated TB patients. IFN-α transcripts were ~3.5 folds higher in TB patients compared to HHC, (p<0.0001). High expression of IFN-α was seen among 46% (56/ 123) of the TB patients and 26%, (22/86) of HHCs. The expression levels of IFN-α correlated with that of IFN transcriptional release factor 7 (IRF) (p<0.0001). In contrast, an inverse relationship exists between PGE2 and IFN-α expression levels; high IFN-α expressers were associated with low levels of PGE2 and vice-versa (Spearman's rho = -0.563; p<0.0001). In-vitro, IFN-α failed to restrict the replication of intracellular M.tb. The anti-mycobacterial activity of IFN-γ was compromised in the presence of IFN-α, but not by IFN-ß. The expression of IFN-α and ß diminished or is absent, among successfully treated TB patients. These observations suggest the utility of assessment of Type I IFNs expression levels as a prognostic marker to monitor tuberculosis patient response to chemotherapy because changes in Type I IFNs expression are expected to precede the clearance and /reduction in bacterial load.


Assuntos
Regulação da Expressão Gênica , Interferon-alfa/metabolismo , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/metabolismo , Estudos de Casos e Controles , Linhagem Celular , Dinoprostona/metabolismo , Humanos , Fatores Reguladores de Interferon/metabolismo , Mycobacterium tuberculosis/crescimento & desenvolvimento , Prognóstico
7.
Science ; 369(6504): 712-717, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32527928

RESUMO

Excessive cytokine signaling frequently exacerbates lung tissue damage during respiratory viral infection. Type I (IFN-α and IFN-ß) and III (IFN-λ) interferons are host-produced antiviral cytokines. Prolonged IFN-α and IFN-ß responses can lead to harmful proinflammatory effects, whereas IFN-λ mainly signals in epithelia, thereby inducing localized antiviral immunity. In this work, we show that IFN signaling interferes with lung repair during influenza recovery in mice, with IFN-λ driving these effects most potently. IFN-induced protein p53 directly reduces epithelial proliferation and differentiation, which increases disease severity and susceptibility to bacterial superinfections. Thus, excessive or prolonged IFN production aggravates viral infection by impairing lung epithelial regeneration. Timing and duration are therefore critical parameters of endogenous IFN action and should be considered carefully for IFN therapeutic strategies against viral infections such as influenza and coronavirus disease 2019 (COVID-19).


Assuntos
Células Epiteliais Alveolares/patologia , Citocinas/metabolismo , Interferon Tipo I/metabolismo , Interferons/metabolismo , Pulmão/patologia , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/patologia , Células Epiteliais Alveolares/imunologia , Animais , Apoptose , Líquido da Lavagem Broncoalveolar/imunologia , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Citocinas/administração & dosagem , Citocinas/imunologia , Feminino , Vírus da Influenza A Subtipo H3N2 , Interferon Tipo I/administração & dosagem , Interferon Tipo I/farmacologia , Interferon-alfa/administração & dosagem , Interferon-alfa/metabolismo , Interferon-alfa/farmacologia , Interferon beta/administração & dosagem , Interferon beta/metabolismo , Interferon beta/farmacologia , Interferons/administração & dosagem , Interferons/farmacologia , Masculino , Camundongos , Infecções por Orthomyxoviridae/metabolismo , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/metabolismo , Receptores de Interferon/genética , Receptores de Interferon/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo
8.
Sci Rep ; 10(1): 8896, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32483133

RESUMO

Initiation of type 1 diabetes (T1D) is marked by the infiltration of plasmacytoid dendritic cells (pDCs) and monocytes in pancreatic islets. Dying beta cells release self-DNA, which forms complexes with antimicrobial peptide, LL37, and its delayed clearance can activate pDCs and monocytes. Here, we studied the phenotypic effects of DNA-LL37 complexes on pDCs and monocytes in 55 recently diagnosed T1D and 25 healthy control (HC) subjects. Following in vitro stimulation with DNA-LL37 complexes, T1D group demonstrated higher frequency and mean fluorescence intensity (MFI) of pDCs expressing IFN-α. Similarly, the monocytes in T1D group showed an increase in MFI of IFN-α. Post-stimulation, an increase in the antigen presentation and co-stimulatory ability of pDCs and monocytes was observed in T1D group, as indicated by higher expression of HLA-DR, CD80 and CD86. Upon co-culture, the stimulated monocytes and pDCs, particularly in the T1D group were able to further activate autologous CD4 + T cells, with increase in expression of CD69 and CD71. Finally, in a transwell assay, the stimulated pDCs and monocytes induced an increase in apoptosis of 1.1B4 beta cells. Additionally, we observed reduced expression of indoleamine 2,3-dioxygenase 1 (IDO1) in pDCs and monocytes of T1D subjects. Our results suggest that DNA-LL37 complexes activate pDCs and monocytes towards a proinflammatory phenotype during pathogenesis of T1D.


Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , DNA/metabolismo , Células Dendríticas/citologia , Diabetes Mellitus Tipo 1/imunologia , Monócitos/citologia , Adolescente , Apresentação do Antígeno , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Estudos de Casos e Controles , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/imunologia , Diabetes Mellitus Tipo 1/metabolismo , Feminino , Humanos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Interferon-alfa/metabolismo , Lectinas Tipo C/metabolismo , Masculino , Monócitos/metabolismo , Receptores da Transferrina/metabolismo , Adulto Jovem
9.
J Virol ; 94(17)2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32581092

RESUMO

Covalently closed circular DNA (cccDNA) of hepadnaviruses exists as an episomal minichromosome in the nucleus of an infected hepatocyte and serves as the template for the transcription of viral mRNAs. It had been demonstrated by others and us that interferon alpha (IFN-α) treatment of hepatocytes induced a prolonged suppression of human and duck hepatitis B virus cccDNA transcription, which is associated with the reduction of cccDNA-associated histone modifications specifying active transcription (H3K9ac or H3K27ac), but not the histone modifications marking constitutive (H3K9me3) or facultative (H3K27me3) heterochromatin formation. In our efforts to identify IFN-induced cellular proteins that mediate the suppression of cccDNA transcription by the cytokine, we found that downregulating the expression of signal transducer and activator of transcription 1 (STAT1), structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1), or promyelocytic leukemia (PML) protein increased basal level of cccDNA transcription activity and partially attenuated IFN-α suppression of cccDNA transcription. In contrast, ectopic expression of STAT1, SMCHD1, or PML significantly reduced cccDNA transcription activity. SMCHD1 is a noncanonical SMC family protein and implicated in epigenetic silencing of gene expression. PML is a component of nuclear domain 10 (ND10) and is involved in suppressing the replication of many DNA viruses. Mechanistic analyses demonstrated that STAT1, SMCHD1, and PML were recruited to cccDNA minichromosomes and phenocopied the IFN-α-induced posttranslational modifications of cccDNA-associated histones. We thus conclude that STAT1, SMCHD1, and PML may partly mediate the suppressive effect of IFN-α on hepadnaviral cccDNA transcription.IMPORTANCE Pegylated IFN-α is the only therapeutic regimen that can induce a functional cure of chronic hepatitis B in a small, but significant, fraction of treated patients. Understanding the mechanisms underlying the antiviral functions of IFN-α in hepadnaviral infection may reveal molecular targets for development of novel antiviral agents to improve the therapeutic efficacy of IFN-α. By a loss-of-function genetic screening of individual IFN-stimulated genes (ISGs) on hepadnaviral mRNAs transcribed from cccDNA, we found that downregulating the expression of STAT1, SMCHD1, or PML significantly increased the level of viral RNAs without altering the level of cccDNA. Mechanistic analyses indicated that those cellular proteins are recruited to cccDNA minichromosomes and induce the posttranslational modifications of cccDNA-associated histones similar to those induced by IFN-α treatment. We have thus identified three IFN-α-induced cellular proteins that suppress cccDNA transcription and may partly mediate IFN-α silencing of hepadnaviral cccDNA transcription.


Assuntos
DNA Circular/metabolismo , Hepadnaviridae/efeitos dos fármacos , Hepadnaviridae/genética , Interferon-alfa/metabolismo , Interferon-alfa/farmacologia , Animais , Antivirais/metabolismo , Antivirais/farmacologia , Linhagem Celular , Galinhas , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , DNA Viral/genética , Epigênese Genética , Infecções por Hepadnaviridae/virologia , Vírus da Hepatite B do Pato/efeitos dos fármacos , Vírus da Hepatite B , Hepatite B Crônica/virologia , Hepatócitos/virologia , Código das Histonas , Histonas/metabolismo , Humanos , Interferon-alfa/genética , Proteína da Leucemia Promielocítica/metabolismo , Processamento de Proteína Pós-Traducional , RNA Viral , Fator de Transcrição STAT1/metabolismo , Transcrição Genética , Replicação Viral
10.
Nat Commun ; 11(1): 2584, 2020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32444635

RESUMO

Interferon-α (IFNα), a type I interferon, is expressed in the islets of type 1 diabetic individuals, and its expression and signaling are regulated by T1D genetic risk variants and viral infections associated with T1D. We presently characterize human beta cell responses to IFNα by combining ATAC-seq, RNA-seq and proteomics assays. The initial response to IFNα is characterized by chromatin remodeling, followed by changes in transcriptional and translational regulation. IFNα induces changes in alternative splicing (AS) and first exon usage, increasing the diversity of transcripts expressed by the beta cells. This, combined with changes observed on protein modification/degradation, ER stress and MHC class I, may expand antigens presented by beta cells to the immune system. Beta cells also up-regulate the checkpoint proteins PDL1 and HLA-E that may exert a protective role against the autoimmune assault. Data mining of the present multi-omics analysis identifies two compound classes that antagonize IFNα effects on human beta cells.


Assuntos
Processamento Alternativo , Células Secretoras de Insulina/fisiologia , Interferon-alfa/metabolismo , Interferon-alfa/farmacologia , Processamento Alternativo/efeitos dos fármacos , Células Cultivadas , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Mineração de Dados , Diabetes Mellitus Tipo 1/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Mapas de Interação de Proteínas , Proteômica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Sítio de Iniciação de Transcrição
11.
Parasitol Res ; 119(5): 1641-1652, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32285266

RESUMO

Clonorchis sinensis (C. sinensis) can induce a food-borne parasitic disease (clonorchiasis). Numerous studies have analyzed functional proteins, immunologic factors, pro-inflammatory cytokines, and cell signaling transduction that promote the development of clonorchiasis. In a previous study, it was shown that C. sinensis adult-derived total protein (CsTP) might be involved in the pathogenesis and development of liver fibrosis via bringing about Th2 immune response. In the present study, further investigation of CsTP on cellular function and inflammatory effect in vitro and in vivo has been elicited. CsTP induced inflammation and autophagy as evidenced by upregulation of TNF-α, IFN-γ, and autophagic markers LC3B and P62. Exposed to CsTP upregulated the antiapoptotic gene Bcl-2 expression, diminished the apoptosis induced by H2O2, but promoted the proliferation and migration of LX-2 cells in proper concentration range. Additionally, the protein levels of p-AKT and p-mTOR were repressed in response to CsTP, suggesting a correlation of blocking the activation of mTOR/AKT signaling pathway. These results revealed that CsTP might exacerbate hepatic pathological changes by regulating cell proliferation, apoptosis, autophagy, and inflammation in the liver and LX-2 cells. Some effects might be partially involved in the mTOR and AKT pathways.


Assuntos
Apoptose/fisiologia , Clonorquíase/patologia , Clonorchis sinensis/patogenicidade , Cirrose Hepática/patologia , Proteínas de Protozoários/metabolismo , Animais , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Clonorquíase/parasitologia , Clonorchis sinensis/genética , Citocinas/metabolismo , Doenças Transmitidas por Alimentos/parasitologia , Humanos , Peróxido de Hidrogênio/metabolismo , Inflamação/patologia , Interferon-alfa/metabolismo , Cirrose Hepática/parasitologia , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima
12.
BMC Biotechnol ; 20(1): 16, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32169063

RESUMO

BACKGROUND: The type I human interferon (IFN) family consists of a group of cytokines with a multiplicity of biological activities, including antiviral, antitumor, and immunomodulatory effects. However, because the half-life of IFN is short, its clinical application is limited. Increasing the yield and biological activity of IFN while extending its half-life is currently the focus of IFN research. RESULTS: Two novel long-acting recombinant human IFN-α2b (rhIFN-α2b) proteins were designed in which the carboxyl-terminal peptide (CTP) of the human chorionic gonadotropin ß su bunit and N-linked glycosylation sequences were linked to rhIFN-α2b. They were designated IFN-1CTPON (fused at the C-terminus of rhIFN-α2b) and IFN-2CTPON (fused at both the C-terminus and N-terminus of rhIFN-α2b). Monoclonal CHO cell strains stably and efficiently expressing the IFNs were successfully selected with methotrexate (MTX), and the highest expression levels were 1468 mg/l and 1196 mg/l for IFN-1CTPON and IFN-2CTPON, respectively. The proteins were purified with affinity chromatography and molecular sieve chromatography. IFN-1CTPON and IFN-2CTPON showed antiviral and antiproliferative activities in vitro. Notably, the half-life of IFN-1CTPON and IFN-2CTPON in vivo were three-fold and two-fold longer than that of commercially available rhIFN-α2b. CONCLUSIONS: CHO cell strains stably expressing long-acting rhIFN-α2b were screened. The purified IFN-CTPON protein has biological activity and an extended half-life, and therefore potential applications.


Assuntos
Antineoplásicos/farmacologia , Antivirais/farmacologia , Gonadotropina Coriônica Humana Subunidade beta/genética , Interferon-alfa/genética , Proteínas Recombinantes de Fusão/farmacologia , Animais , Células CHO , Proliferação de Células/efeitos dos fármacos , Gonadotropina Coriônica Humana Subunidade beta/química , Cromatografia de Afinidade , Cricetulus , Preparações de Ação Retardada , Glicosilação , Meia-Vida , Células HeLa , Vírus da Hepatite B/efeitos dos fármacos , Humanos , Interferon-alfa/metabolismo , Interferon-alfa/farmacologia , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/metabolismo
13.
J Virol ; 94(11)2020 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-32188736

RESUMO

Hepatitis B virus (HBV) chronically infects approximately 350 million people worldwide, and 600,000 deaths are caused by HBV-related hepatic failure, liver cirrhosis, and hepatocellular carcinoma annually. It is important to reveal the mechanism underlying the regulation of HBV replication. This study demonstrated that osteopetrosis-associated transmembrane protein 1 (Ostm1) plays an inhibitory role in HBV replication. Ostm1 represses the levels of HBeAg and HBsAg proteins, HBV 3.5-kb and 2.4/2.1-kb RNAs, and core-associated DNA in HepG2, Huh7, and NTCP-HepG2 cells. Notably, Ostm1 has no direct effect on the activity of HBV promoters or the transcription of HBV RNAs; instead, Ostm1 binds to HBV RNA to facilitate RNA decay. Detailed studies further demonstrated that Ostm1 binds to and recruits the RNA exosome complex to promote the degradation of HBV RNAs, and knockdown of the RNA exosome component exonuclease 3 (Exosc3) leads to the elimination of Ostm1-mediated repression of HBV replication. Mutant analyses revealed that the N-terminal domain, the transmembrane domain, and the C-terminal domain are responsible for the repression of HBV replication, and the C-terminal domain is required for interaction with the RNA exosome complex. Moreover, Ostm1 production is not regulated by interferon-α (IFN-α) or IFN-γ, and the expression of IFN signaling components is not affected by Ostm1, suggesting that Ostm1 anti-HBV activity is independent of the IFN signaling pathway. In conclusion, this study revealed a distinct mechanism underlying the repression of HBV replication, in which Ostm1 binds to HBV RNA and recruits RNA exosomes to degrade viral RNA, thereby restricting HBV replication.IMPORTANCE Hepatitis B virus (HBV) is a human pathogen infecting the liver to cause a variety of diseases ranging from acute hepatitis to advanced liver diseases, fulminate hepatitis, liver cirrhosis, and hepatocellular carcinoma, thereby causing a major health problem worldwide. In this study, we demonstrated that Ostm1 plays an inhibitory role in HBV protein production, RNA expression, and DNA replication. However, Ostm1 has no effect on the activities of the four HBV promoters; instead, it binds to HBV RNA and recruits RNA exosomes to promote HBV RNA degradation. We further demonstrated that the anti-HBV activity of Ostm1 is independent of the interferon signaling pathway. In conclusion, this study reveals a distinct mechanism underlying the repression of HBV replication and suggests that Ostm1 is a potential therapeutic agent for HBV infection.


Assuntos
Exossomos/metabolismo , Vírus da Hepatite B/fisiologia , Proteínas de Membrana/metabolismo , RNA Viral/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Replicação Viral , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Exossomos/genética , Exossomos/virologia , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Células Hep G2 , Antígenos de Superfície da Hepatite B/genética , Antígenos de Superfície da Hepatite B/metabolismo , Antígenos E da Hepatite B/genética , Antígenos E da Hepatite B/metabolismo , Humanos , Interferon-alfa/genética , Interferon-alfa/metabolismo , Interferon gama/genética , Interferon gama/metabolismo , Proteínas de Membrana/genética , Domínios Proteicos , RNA Viral/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ubiquitina-Proteína Ligases/genética
14.
Int J Mol Sci ; 21(3)2020 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-32046242

RESUMO

Among environmental factors likely associated with Alzheimer's disease (AD), persistent virus infections, and age-related progressive decline of immune competence might play a pivotal role. However, AD antimicrobial brain immune responses are poorly investigated. The present study focused on genes involved in antimicrobial defenses, especially against virus infections, in the AD brain. In particular, mRNA levels of IRF7, MED23, IL28B, and IFN-α genes were analyzed in hippocampus and temporal cortex brain samples from AD and non-demented controls. All subjects were also genotyped for APOE ε, IRF7, MED23, and IL28B gene polymorphisms. Most AD patients showed decreased mRNA levels of all investigated genes in the hippocampus and temporal cortex. However, a small group of AD patients showed increased hippocampal mRNA expression of MED23, IL28B, and IFN-α. mRNA levels of MED23, IL28B, IFN-α from the hippocampus and those of MED23 from the temporal cortex were further decreased in APOE ε4 allele AD carriers. Moreover, rs6598008 polymorphism of IRF7 was significantly associated with decreased hippocampal expression of IRF7, MED23, IL28B, and IFN-α. These findings suggest that AD brains show impaired innate antimicrobial gene expression profiles, and individual genetic makeup, such as positivity for the APOE ε4 and IRF7 A alleles, might affect brain immune efficiency.


Assuntos
Doença de Alzheimer/imunologia , Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Hipocampo/metabolismo , Imunidade Inata/fisiologia , Idoso , Idoso de 80 Anos ou mais , Alelos , Doença de Alzheimer/genética , Feminino , Humanos , Imunidade Inata/genética , Fator Regulador 7 de Interferon/genética , Fator Regulador 7 de Interferon/metabolismo , Interferon-alfa/genética , Interferon-alfa/metabolismo , Interferons/genética , Interferons/metabolismo , Masculino , Complexo Mediador/genética , Complexo Mediador/metabolismo , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
15.
Scand J Immunol ; 91(5): e12863, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31909839

RESUMO

Interferon α (IFNα) is a cytokine that mediates diverse immune responses to tumours. It is the oldest immune-based oncologic drug and has been widely used to treat various malignancies in humans. Yet, the use of IFNα in cancer therapy has only resulted in limited success and even led to worse clinical outcomes under certain instances. The emergence of the cancer immunoediting concept-which implicates the host immune system in promoting tumour growth-recapitulates the need to evaluate the immune functions of IFNα. This review proposes that IFNα has dual opposing roles in cancer development based on the mutational status of its signalling components, which determines the expression of anti- or pro-tumorigenic IFN-stimulated genes (ISGs). This duality may translate into new applications of IFNα in cancer immunotherapy.


Assuntos
Imunomodulação , Interferon-alfa/metabolismo , Neoplasias/imunologia , Neoplasias/metabolismo , Evasão Tumoral , Animais , Terapia Combinada , Gerenciamento Clínico , Suscetibilidade a Doenças , Humanos , Imunoterapia , Neoplasias/etiologia , Neoplasias/terapia
16.
Immunol Cell Biol ; 98(3): 203-214, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31916630

RESUMO

The NZB/W F1 (F1) mice develop severe disease that is similar to human systemic lupus erythematosus. By contrast, each parent strain, NZB or NZW, has limited autoimmunity, suggesting traits of both strains contribute to pathogenesis. Although many of the contributing genes have been identified, the contributing cellular abnormality associated with each parent strain remains unresolved. Given that plasmacytoid dendritic cells (pDCs) are key to the pathogenesis of lupus, we investigated the properties of pDCs from NZB and NZW mice. We found that NZB mouse had higher numbers of pDCs, with much of the increase being contributed by a more abundant CD8+ pDC subset. This was associated with prolonged survival and stronger proliferation of CD4+ T cells. By contrast, NZW pDCs had heightened capacity to produce interferon-α (IFNα) and IFNλ, and promoted stronger B-cell proliferation upon CpG stimulation. Thus, our data reveal the different functional and numerical characteristics of pDCs from NZW and NZB mouse.


Assuntos
Autoimunidade , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Ativação Linfocitária/imunologia , Animais , Linfócitos B/imunologia , Antígenos CD8/imunologia , Antígenos CD8/metabolismo , Sobrevivência Celular/imunologia , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Fatores de Transcrição Forkhead/metabolismo , Interferon-alfa/metabolismo , Interferons/metabolismo , Lúpus Eritematoso Sistêmico/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NZB , Camundongos Knockout , Camundongos Transgênicos , Oligodesoxirribonucleotídeos/farmacologia
17.
Immunol Cell Biol ; 98(3): 215-228, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31919905

RESUMO

Epicutaneous (EC) sensitization with protein allergens is the most important sensitization route for atopic dermatitis. Plasmacytoid dendritic cells (pDCs) are characterized by massive secretion of interferon-α (IFNα). B6 mice are T helper type 1 (Th1)-prone and are representative of non-atopic humans, whereas BALB/c mice are Th2-prone and are representative of atopic humans. Here, we show that naïve BALB/c mice contain a greater number of nonactivated pDCs in peripheral lymph nodes (LNs) than do naïve B6 mice. Naïve BALB/c mice also have more of the CD8α- subset in LNs than naïve B6 mice. Moreover, in vivo depletion of pDCs during EC sensitization results in enhanced Th2 responses in BALB/c mice, but not in B6 mice. Mechanistically, when BALB/c mice undergo EC sensitization, there is an increase in pDCs entering draining LNs. These cells exhibit modest activation including comparable costimulation expression but increased cytokine expression compared with those of naïve mice. In vivo depletion of pDCs during EC sensitization significantly increases the activation of dermal dendritic cells (dDCs) suggesting a regulatory effect on these cells. To this end, a suppressive effect of pDCs on conventional dendritic cells was also demonstrated in vitro. Further, in vivo blockade of IFNα by an anti-IFNAR antibody (Ab) or in vivo reduction of IFNα production of pDCs by anti-siglec-H Ab both resulted in enhanced activation of dDCs. Collectively, our results demonstrate that pDCs suppress Th2 responses induced by EC sensitization via IFNα-mediated regulation of dDCs.


Assuntos
Células Dendríticas/imunologia , Dermatite Atópica/imunologia , Interferon-alfa/metabolismo , Pele/imunologia , Células Th2/imunologia , Animais , Células Dendríticas/metabolismo , Feminino , Imunização , Interleucinas/metabolismo , Linfonodos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Pele/patologia
19.
J Dermatol ; 47(2): 104-113, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31833093

RESUMO

Psoriasis is a chronic skin inflammatory disorder, the immune mechanism of which has been profoundly elucidated in the past few years. The dominance of the interleukin (IL)-23/IL-17 axis is a significant breakthrough in the understanding of the pathogenesis of psoriasis, and treatment targeting IL-23 and IL-17 has successfully benefited patients with the disease. The skin contains a complex network of dendritic cells (DC) mainly composed of epidermal Langerhans cells, bone marrow-derived dermal conventional DC, plasmacytoid DC and inflammatory DC. As the prominent cellular source of α-interferon, tumor necrosis factor-α, IL-12 and IL-23, DC play a pivotal role in psoriasis. Thus, targeting pathogenic DC subsets is a valid strategy for alleviating and preventing psoriasis and other DC-derived diseases. In this review, we survey the known role of DC in this disease.


Assuntos
Células Dendríticas/imunologia , Psoríase/imunologia , Animais , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Fármacos Dermatológicos/farmacologia , Fármacos Dermatológicos/uso terapêutico , Modelos Animais de Doenças , Humanos , Interferon-alfa/metabolismo , Interleucina-17/antagonistas & inibidores , Interleucina-17/metabolismo , Interleucina-23/antagonistas & inibidores , Interleucina-23/metabolismo , Psoríase/tratamento farmacológico , Psoríase/patologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Pele/citologia , Pele/imunologia , Pele/patologia , Fator de Necrose Tumoral alfa/metabolismo
20.
J Immunol ; 204(2): 348-359, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31826941

RESUMO

Autoimmunity can result when cells fail to properly dispose of DNA. Mutations in the three-prime repair exonuclease 1 (TREX1) cause a spectrum of human autoimmune diseases resembling systemic lupus erythematosus. The cytosolic dsDNA sensor, cyclic GMP-AMP synthase (cGAS), and the stimulator of IFN genes (STING) are required for pathogenesis, but specific cells in which DNA sensing and subsequent type I IFN (IFN-I) production occur remain elusive. In this study, we demonstrate that TREX1 D18N catalytic deficiency causes dysregulated IFN-I signaling and autoimmunity in mice. Moreover, we show that bone marrow-derived cells drive this process. We identify both innate immune and, surprisingly, activated T cells as sources of pathological IFN-α production. These findings demonstrate that TREX1 enzymatic activity is crucial to prevent inappropriate DNA sensing and IFN-I production in immune cells, including normally low-level IFN-α-producing cells. These results expand our understanding of DNA sensing and innate immunity in T cells and may have relevance to the pathogenesis of human disease caused by TREX1 mutation.


Assuntos
Exodesoxirribonucleases/genética , Lúpus Eritematoso Sistêmico/genética , Fosfoproteínas/genética , Linfócitos T/imunologia , Animais , Autoantígenos/imunologia , Autoimunidade , Células Cultivadas , DNA/imunologia , Modelos Animais de Doenças , Humanos , Imunidade Inata , Interferon-alfa/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nucleotidiltransferases/metabolismo
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