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1.
Immunity ; 51(3): 451-464.e6, 2019 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-31471108

RESUMO

Type I and III interferons (IFNs) activate similar downstream signaling cascades, but unlike type I IFNs, type III IFNs (IFNλ) do not elicit strong inflammatory responses in vivo. Here, we examined the molecular mechanisms underlying this disparity. Type I and III IFNs displayed kinetic differences in expression of IFN-stimulated genes and proinflammatory responses, with type I IFNs preferentially stimulating expression of the transcription factor IRF1. Type III IFNs failed to induce IRF1 expression because of low IFNλ receptor abundance and insufficient STAT1 activation on epithelial cells and thus did not activate the IRF1 proinflammatory gene program. Rather, IFNλ stimulation preferentially induced factors implicated in tissue repair. Our findings suggest that IFN receptor compartmentalization and abundance confer a spatiotemporal division of labor where type III IFNs control viral spread at the site of the infection while restricting tissue damage; the transient induction of inflammatory responses by type I IFNs recruits immune effectors to promote protective immunity.


Assuntos
Fator Regulador 1 de Interferon/imunologia , Interferon Tipo I/imunologia , Interferons/imunologia , Animais , Linhagem Celular , Células Epiteliais/imunologia , Humanos , Inflamação/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator de Transcrição STAT1/imunologia
2.
Cells ; 8(7)2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31269695

RESUMO

Background: The aim of the study was to investigate the intra-host variability through next-generation-sequencing (NGS) of the NS5A-gene in nosocomial transmission-clusters observed in two Italian hospitals among hepatitis C virus (HCV)-genotype-1b infected patients. Methods: HCV-sequencing was performed by Sanger-sequencing (NS3 + NS5A + NS5B) and by NGS (NS5A, MiSeq-Illumina) in 15 HCV-1b infected patients [five acute with onco-hematologic-disease and 10 (4/6 acute/chronic) with ß-thalassemia]. Resistance-associated-substitutions (RAS) were analysed by Geno2pheno-algorithm. Nucleotide-sequence-variability (NSV, at 1%, 2%, 5%, 10% and 15% NGS-cutoffs) and Shannon entropy were estimated. Phylogenetic analysis was performed by Mega6-software and Bayesian-analysis. Results: Phylogenetic analysis showed five transmission-clusters: one involving four HCV-acute onco-hematologic-patients; one involving three HCV-chronic ß-thalassemia-patients and three involving both HCV-acute and chronic ß-thalassemia-patients. The NS5A-RAS Y93H was found in seven patients, distributed differently among chronic/acute patients involved in the same transmission-clusters, independently from the host-genetic IL-28-polymorphism. The intra-host NSV was higher in chronic-patients versus acute-patients, at all cutoffs analyzed (p < 0.05). Even though Shannon-entropy was higher in chronic-patients, significantly higher values were observed only in chronic ß-thalassemia-patients versus acute ß-thalassemia-patients (p = 0.01). Conclusions: In nosocomial HCV transmission-clusters, the intra-host HCV quasispecies divergence in patients with acute-infection was very low in comparison to that in chronic-infection. The NS5A-RAS Y93H was often transmitted and distributed differently within the same transmission-clusters, independently from the IL-28-polymorphism.


Assuntos
Infecção Hospitalar/virologia , Hepacivirus/genética , Hepatite C/virologia , Proteínas não Estruturais Virais/genética , Talassemia beta/terapia , Doença Aguda , Adulto , Substituição de Aminoácidos , Antivirais/farmacologia , Antivirais/uso terapêutico , Transfusão de Sangue , Doença Crônica , Infecção Hospitalar/tratamento farmacológico , Infecção Hospitalar/transmissão , Farmacorresistência Viral/genética , Feminino , Genótipo , Hepacivirus/patogenicidade , Hepatite C/tratamento farmacológico , Hepatite C/transmissão , Sequenciamento de Nucleotídeos em Larga Escala , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Interferons/genética , Interferons/imunologia , Masculino , Pessoa de Meia-Idade , Filogenia , Polimorfismo de Nucleotídeo Único , Talassemia beta/genética , Talassemia beta/imunologia
3.
Nat Commun ; 10(1): 2230, 2019 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-31110180

RESUMO

LNK (SH2B3) is a key negative regulator of JAK-STAT signaling which has been extensively studied in malignant hematopoietic diseases. We found that LNK is significantly elevated in cutaneous melanoma; this elevation is correlated with hyperactive signaling of the RAS-RAF-MEK pathway. Elevated LNK enhances cell growth and survival in adverse conditions. Forced expression of LNK inhibits signaling by interferon-STAT1 and suppresses interferon (IFN) induced cell cycle arrest and cell apoptosis. In contrast, silencing LNK expression by either shRNA or CRISPR-Cas9 potentiates the killing effect of IFN. The IFN-LNK signaling is tightly regulated by a negative feedback mechanism; melanoma cells exposed to IFN upregulate expression of LNK to prevent overactivation of this signaling pathway. Our study reveals an unappreciated function of LNK in melanoma and highlights the critical role of the IFN-STAT1-LNK signaling axis in this potentially devastating disease. LNK may be further explored as a potential therapeutic target for melanoma immunotherapy.


Assuntos
Interferons/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Melanoma/patologia , Proteínas/metabolismo , Neoplasias Cutâneas/patologia , Animais , Apoptose , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Células HEK293 , Humanos , Interferons/imunologia , Melanoma/imunologia , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Fator de Transcrição STAT1/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Nat Commun ; 10(1): 2261, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-31113940

RESUMO

Cyclic GMP-AMP synthase (cGAS) is the primary sensor for aberrant intracellular dsDNA producing the cyclic dinucleotide cGAMP, a second messenger initiating cytokine production in subsets of myeloid lineage cell types. Therefore, inhibition of the enzyme cGAS may act anti-inflammatory. Here we report the discovery of human-cGAS-specific small-molecule inhibitors by high-throughput screening and the targeted medicinal chemistry optimization for two molecular scaffolds. Lead compounds from one scaffold co-crystallize with human cGAS and occupy the ATP- and GTP-binding active site. The specificity and potency of these drug candidates is further documented in human myeloid cells including primary macrophages. These novel cGAS inhibitors with cell-based activity will serve as probes into cGAS-dependent innate immune pathways and warrant future pharmacological studies for treatment of cGAS-dependent inflammatory diseases.


Assuntos
Descoberta de Drogas/métodos , Inibidores Enzimáticos/farmacologia , Nucleotidiltransferases/antagonistas & inibidores , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Células Cultivadas , Cristalografia por Raios X , DNA/imunologia , DNA/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Ensaios de Triagem em Larga Escala/métodos , Humanos , Imunidade Inata/efeitos dos fármacos , Interferons/imunologia , Interferons/metabolismo , Macrófagos , Modelos Moleculares , Nucleotídeos Cíclicos/imunologia , Nucleotídeos Cíclicos/metabolismo , Nucleotidiltransferases/imunologia , Nucleotidiltransferases/isolamento & purificação , Nucleotidiltransferases/metabolismo , Cultura Primária de Células , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
5.
Biomed Res Int ; 2019: 4518163, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31008105

RESUMO

Inactivation of rabies virus is essential for rabies vaccine preparation where the inactivating compound that is currently recommended for rabies vaccine preparation is ß-propiolactone (ß-PL). This compound is considered better than phenol and formalin but it is expensive and potentially carcinogenic. Data revealed that Ascorbic acid (AA) with cupric ions could yield complete and irreversible inactivation of rabies virus without adversely affecting its antigenicity. Additionally, the results of testing the vaccine potency with the selected inactivating compounds were comparable (P<0.05), and ED50 was higher than the recommended World Health Organization (WHO) limits. The use of HemaGel (plasma substitute) for testing vaccine stabilization was compared with the currently used vaccine stabilizers (human albumin and lactose). HemaGel yielded better stability than the other tested stabilizers. Monitoring of cellular and humoral immune responses indicated that both the total IgG level against rabies vaccine and the IFN and IL5 levels obtained with the HemaGel-stabilized vaccines were higher than those obtained with human albumin- and lactose-stabilized vaccine candidates.


Assuntos
Imunogenicidade da Vacina/efeitos dos fármacos , Propiolactona/farmacologia , Vacinas Antirrábicas/farmacologia , Raiva/prevenção & controle , Albuminas/farmacologia , Animais , Anticorpos Antivirais/efeitos dos fármacos , Anticorpos Antivirais/imunologia , Ácido Ascórbico/farmacologia , Humanos , Imunoglobulina G/imunologia , Interferons/imunologia , Interleucina-5 , Lactose/química , Propiolactona/química , Raiva/imunologia , Raiva/virologia , Vacinas Antirrábicas/química , Vacinas Antirrábicas/genética , Vacinas Antirrábicas/imunologia , Vírus da Raiva/imunologia , Vírus da Raiva/patogenicidade , Potência de Vacina , Células Vero/virologia
6.
Biomed J ; 42(1): 19-26, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30987701

RESUMO

The role of host genetics in influenza infection is unclear despite decades of interest. Confounding factors such as age, sex, ethnicity and environmental factors have made it difficult to assess the role of genetics without influence. In recent years a single nucleotide polymorphism, interferon-induced transmembrane protein 3 (IFITM3) rs12252, has been shown to alter the severity of influenza infection in Asian populations. In this review we investigate this polymorphism as well as several others suggested to alter the host's defence against influenza infection. In addition, we highlight the open questions surrounding the viral restriction protein IFITM3 with the hope that by answering some of these questions we can elucidate the mechanism of IFITM3 viral restriction and therefore how this restriction is altered due to the rs12252 polymorphism.


Assuntos
Predisposição Genética para Doença/genética , Influenza Humana/genética , Interferons/genética , Proteínas de Membrana/genética , Proteínas de Ligação a RNA/genética , Genótipo , Humanos , Influenza Humana/imunologia , Interferons/imunologia , Proteínas de Membrana/imunologia , Proteínas de Ligação a RNA/imunologia
7.
Immunity ; 50(4): 907-923, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30995506

RESUMO

Type I interferons (IFNs) (IFN-α, IFN-ß) and type III IFNs (IFN-λ) share many properties, including induction by viral infection, activation of shared signaling pathways, and transcriptional programs. However, recent discoveries have revealed context-specific functional differences. Here, we provide a comprehensive review of type I and type III IFN activities, highlighting shared and distinct features from molecular mechanisms through physiological responses. Beyond discussing canonical antiviral functions, we consider the adaptive immune priming, anti-tumor, and autoimmune functions of IFNs. We discuss a model wherein type III IFNs serve as a front-line defense that controls infection at epithelial barriers while minimizing damaging inflammatory responses, reserving the more potent type I IFN response for when local responses are insufficient. In this context, we discuss current therapeutic applications targeting these cytokine pathways and highlight gaps in understanding of the biology of type I and type III IFNs in health and disease.


Assuntos
Interferon Tipo I/imunologia , Interferons/imunologia , Imunidade Adaptativa , Animais , Antivirais/uso terapêutico , Doenças Autoimunes/etiologia , Doenças Autoimunes/imunologia , Células Epiteliais/imunologia , Feminino , Humanos , Interferon Tipo I/efeitos adversos , Interferon Tipo I/uso terapêutico , Interferons/efeitos adversos , Interferons/uso terapêutico , Masculino , Troca Materno-Fetal/imunologia , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Especificidade de Órgãos , Gravidez , Transdução de Sinais/imunologia , Transcrição Genética , Transcriptoma , Viroses/tratamento farmacológico , Viroses/imunologia
8.
Immunity ; 50(4): 975-991, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30995510

RESUMO

Asthma is a chronic inflammatory airway disease associated with type 2 cytokines interleukin-4 (IL-4), IL-5, and IL-13, which promote airway eosinophilia, mucus overproduction, bronchial hyperresponsiveness (BHR), and immunogloubulin E (IgE) synthesis. However, only half of asthma patients exhibit signs of an exacerbated Type 2 response. "Type 2-low" asthma has different immune features: airway neutrophilia, obesity-related systemic inflammation, or in some cases, few signs of immune activation. Here, we review the cytokine networks driving asthma, placing these in cellular context and incorporating insights from cytokine-targeting therapies in the clinic. We discuss established and emerging paradigms in the context of the growing appreciation of disease heterogeneity and argue that the development of new and improved therapeutics will require understanding the diverse mechanisms underlying the spectrum of asthma pathologies.


Assuntos
Asma/imunologia , Citocinas/imunologia , Imunidade Adaptativa , Corticosteroides/uso terapêutico , Alérgenos/imunologia , Animais , Antiasmáticos/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Asma/classificação , Asma/tratamento farmacológico , Asma/fisiopatologia , Ensaios Clínicos como Assunto , Citocinas/antagonistas & inibidores , Células Epiteliais/imunologia , Humanos , Inflamação/imunologia , Interferons/imunologia , Camundongos , Camundongos Knockout , Modelos Imunológicos , Células Th2/imunologia
9.
Nat Microbiol ; 4(6): 914-924, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30936491

RESUMO

Interferon (IFN)-mediated antiviral responses are central to host defence against viral infection. Despite the existence of at least 20 IFNs, there are only three known cell surface receptors. IFN signalling and viral evasion mechanisms form an immensely complex network that differs across species. In this Review, we begin by highlighting some of the advances that have been made towards understanding the complexity of differential IFN signalling inputs and outputs that contribute to antiviral defences. Next, we explore some of the ways viruses can interfere with, or circumvent, these defences. Lastly, we address the largely under-reviewed impact of IFN signalling on host tropism, and we offer perspectives on the future of research into IFN signalling complexity and viral evasion across species.


Assuntos
Evasão da Resposta Imune/imunologia , Interferon Tipo I/imunologia , Interferons/imunologia , Transdução de Sinais/imunologia , Viroses/imunologia , Animais , Antivirais , Regulação da Expressão Gênica/imunologia , Regulação da Expressão Gênica/fisiologia , Interações Hospedeiro-Patógeno/imunologia , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Evasão da Resposta Imune/fisiologia , Imunidade Inata/imunologia , Imunidade Inata/fisiologia , Interferon Tipo I/genética , Interferon Tipo I/fisiologia , Interferons/genética , Interferons/fisiologia , Transdução de Sinais/fisiologia , Tropismo , Vírus/imunologia
10.
Fish Shellfish Immunol ; 89: 76-82, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30917925

RESUMO

Transducin ß-like 1 X-linked receptor 1 (TBLR1) was identified as an important component of nuclear receptor corepressor (N-CoR) complex, and functionally participated in regulation of transcriptional activation. However, the potential roles of TBLR1 in innate immune response still remain uncertain. In the present work, a novel TBLR1 from orange-spotted grouper, Epinephelus coioides (named as EcTBLR1) was cloned and its effect on fish virus infection was characterized. The full length open reading frame (ORF) of EcTBLR1 was 1548 bp and encoded a putative 515-aa polypeptide, which shared 99% and 95% identity with its homologue from large yellow croaker (Larimichthys crocea) and human (Homo sapiens), respectively. Quantitative PCR (qPCR) analysis revealed a ubiquitous expression of EcTBLR1 in different tissues with remarkable expression in brain, spleen and head-kidney. Subcellular location analysis showed that EcTBLR1 was mainly located in cytoplasm of grouper spleen cells, and partly translocated into nucleus after infection with red spotted grouper nervous necrosis virus (RGNNV). Moreover, RGNNV infection suppressed the protein synthesis of EcTBLR1 in grouper cells. Using RNA interference (RNAi) technology, we found that effective knock-down of EcTBLR1 significantly suppressed the transcription of RGNNV capsid protein (Cp) and RNA-dependent RNA polymerase (RdRp) genes, which implied the crucial role of EcTBLR1 in RGNNV infection. Consistently, overexpression of EcTBLR1 in vitro significantly inhibited IFN promoter activity, as well as the transcription of IFN-related downstream effectors, including interferon stimulated gene 15 (ISG15) and interferon regulatory factor 3 (IRF3). Together, our results for the first time demonstrated that fish TBLR1 might exert critical roles during fish RNA virus replication by negatively regulating interferon response.


Assuntos
Bass/genética , Bass/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Proteínas Repressoras/genética , Proteínas Repressoras/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Interferons/imunologia , Nodaviridae/fisiologia , Filogenia , Infecções por Vírus de RNA/imunologia , Infecções por Vírus de RNA/veterinária , Proteínas Repressoras/química , Alinhamento de Sequência/veterinária
11.
Nature ; 567(7747): 262-266, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30842662

RESUMO

Cyclic GMP-AMP (cGAMP) synthase (cGAS) detects infections or tissue damage by binding to microbial or self DNA in the cytoplasm1. Upon binding DNA, cGAS produces cGAMP that binds to and activates the adaptor protein STING, which then activates the kinases IKK and TBK1 to induce interferons and other cytokines2-6. Here we report that STING also activates autophagy through a mechanism that is independent of TBK1 activation and interferon induction. Upon binding cGAMP, STING translocates to the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) and the Golgi in a process that is dependent on the COP-II complex and ARF GTPases. STING-containing ERGIC serves as a membrane source for LC3 lipidation, which is a key step in autophagosome biogenesis. cGAMP induced LC3 lipidation through a pathway that is dependent on WIPI2 and ATG5 but independent of the ULK and VPS34-beclin kinase complexes. Furthermore, we show that cGAMP-induced autophagy is important for the clearance of DNA and viruses in the cytosol. Interestingly, STING from the sea anemone Nematostella vectensis induces autophagy but not interferons in response to stimulation by cGAMP, which suggests that induction of autophagy is a primordial function of the cGAS-STING pathway.


Assuntos
Autofagia , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/metabolismo , Transdução de Sinais , Animais , Autofagossomos/metabolismo , Proteína 5 Relacionada à Autofagia/deficiência , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/deficiência , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteína Beclina-1/deficiência , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Citosol/virologia , Vírus de DNA/genética , Vírus de DNA/metabolismo , DNA Viral/metabolismo , Retículo Endoplasmático/metabolismo , Evolução Molecular , Complexo de Golgi/metabolismo , Células HEK293 , Humanos , Interferons/biossíntese , Interferons/imunologia , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Nucleotídeos Cíclicos/imunologia , Nucleotídeos Cíclicos/metabolismo , Proteínas de Ligação a Fosfato , Transporte Proteico , Proteínas Serina-Treonina Quinases/metabolismo , Anêmonas-do-Mar , Proteínas de Transporte Vesicular/metabolismo
12.
Cell Host Microbe ; 25(4): 602-616.e7, 2019 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-30902577

RESUMO

Establishing the balance between positive and negative innate immune mechanisms is crucial for maintaining homeostasis. Here we uncover the regulatory crosstalk between two previously unlinked innate immune receptor families: RIG-I, an anti-viral cytosolic receptor activated type I interferon production, and NLR (nucleotide-binding domain, leucine repeat domain-containing protein). We show that NLRP12 dampens RIG-I-mediated immune signaling against RNA viruses by controlling RIG-I's association with its adaptor MAVS. The nucleotide-binding domain of NLRP12 interacts with the ubiquitin ligase TRIM25 to prevent TRIM25-mediated, Lys63-linked ubiquitination and activation of RIG-I. NLRP12 also enhances RNF125-mediated, Lys48-linked degradative ubiquitination of RIG-I. Vesicular stomatitis virus (VSV) infection downregulates NLRP12 expression to allow RIG-I activation. Myeloid-cell-specific Nlrp12-deficient mice display a heightened interferon and TNF response and are more resistant to VSV infection. These results indicate that NLRP12 functions as a checkpoint for anti-viral RIG-I activation.


Assuntos
Proteína DEAD-box 58/imunologia , Proteínas de Ligação a DNA/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Infecções por Vírus de RNA/imunologia , Vírus de RNA/fisiologia , Fatores de Transcrição/imunologia , Animais , Proteína DEAD-box 58/genética , Proteínas de Ligação a DNA/genética , Feminino , Humanos , Interferons/genética , Interferons/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Infecções por Vírus de RNA/genética , Infecções por Vírus de RNA/virologia , Vírus de RNA/genética , Fatores de Transcrição/genética , Ubiquitinação
13.
Science ; 363(6434)2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30923196

RESUMO

Bacteriophage are abundant at sites of bacterial infection, but their effects on mammalian hosts are unclear. We have identified pathogenic roles for filamentous Pf bacteriophage produced by Pseudomonas aeruginosa (Pa) in suppression of immunity against bacterial infection. Pf promote Pa wound infection in mice and are associated with chronic human Pa wound infections. Murine and human leukocytes endocytose Pf, and internalization of this single-stranded DNA virus results in phage RNA production. This triggers Toll-like receptor 3 (TLR3)- and TIR domain-containing adapter-inducing interferon-ß (TRIF)-dependent type I interferon production, inhibition of tumor necrosis factor (TNF), and the suppression of phagocytosis. Conversely, immunization of mice against Pf prevents Pa wound infection. Thus, Pf triggers maladaptive innate viral pattern-recognition responses, which impair bacterial clearance. Vaccination against phage virions represents a potential strategy to prevent bacterial infection.


Assuntos
Tolerância Imunológica , Fagocitose/imunologia , Infecções por Pseudomonas/imunologia , Fagos de Pseudomonas/fisiologia , Pseudomonas aeruginosa/patogenicidade , Pseudomonas aeruginosa/virologia , Infecção dos Ferimentos/imunologia , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/imunologia , Animais , Anticorpos Antivirais/imunologia , Humanos , Interferons/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Fagos de Pseudomonas/imunologia , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/imunologia , Fator de Necrose Tumoral alfa/metabolismo
14.
Res Vet Sci ; 124: 166-177, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30903969

RESUMO

Sungri/96 vaccine strain is considered the most potent vaccine providing long-term immunity against peste des petits ruminants (PPR) in India. Previous studies in our laboratory highlighted induction of robust antiviral response in an interferon independent manner at 48 h and 120 h post infection (p.i.). However, immune response at the earliest time point 6 h p.i. (time taken to complete one PPRV life cycle), in PBMCs infected with Sungri/96 vaccine virus has not been investigated. This study was taken up to understand the global gene expression profiling of goat PBMCs after Sungri/96 PPRV vaccine strain infection at 6 h post infection (p.i.). A total of 1926 differentially expressed genes (DEGs) were identified with 616 - upregulated and 1310 - downregulated. TLR7/TLR3, IRF7/IRF1, ISG20, IFIT1/IFIT2, IFITM3, IL27 and TREX1 were identified as key immune sensors and antiviral candidate genes. Interestingly, type I interferons (IFNα/ß) were not differentially expressed at this time point as well. TREX1, an exonuclease which inhibits type I interferons at the early stage of virus infection was found to be highly upregulated. IL27, an important antiviral host immune factor was significantly upregulated. ISG20, an antiviral interferon induced gene with exonuclease activity specific to ssRNA viruses was highly expressed. Functional profiling of DEGs showed significant enrichment of immune system processes with 233 genes indicating initiation of immune defense response in host cells. Protein interaction network showed important innate immune molecules in the immune network with high connectivity. The study highlights important immune and antiviral genes at the earliest time point.


Assuntos
Doenças das Cabras/prevenção & controle , Imunidade Inata/genética , Leucócitos Mononucleares/imunologia , Peste dos Pequenos Ruminantes/imunologia , Transcriptoma/imunologia , Vacinas Virais/imunologia , Animais , Antivirais/imunologia , Perfilação da Expressão Gênica/veterinária , Doenças das Cabras/imunologia , Cabras , Índia , Interferons/imunologia , Leucócitos Mononucleares/virologia , Vírus da Peste dos Pequenos Ruminantes/imunologia
15.
J Immunol ; 202(8): 2407-2420, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30850476

RESUMO

In mammals, tripartite motif (TRIM) proteins have emerged as pivotal players endowed with, directly, antiviral effects and, indirectly, modulatory capacity of the innate immune response. An unprecedented expansion of TRIM family has occurred in fish; however, the functional role of fish TRIM family members remains largely unknown. In this study, we identify a species-specific TRIM gene from crucian carp Carassius auratus, named FTRCA1, phylogenetically similar to the members of finTRIM, a subfamily of TRIM exclusively in teleost fish. FTRCA1 is induced by IFN and IFN stimuli as a typical IFN-stimulated gene. Overexpression of FTRCA1 negatively regulates IFN antiviral response by inhibition of IRF3 phosphorylation; consistently, knockdown of FTRCA1 results in enhanced levels of IRF3 phosphorylation and also IFN expression following poly(I:C) transfection. Whereas FTRCA1 is associated with several pivotal signaling molecules of RIG-I-like receptor pathway, its association with TBK1 results in autophage-lysosomal degradation of TBK1, thus abrogating the downstream IFN induction. Interestingly, FTRCA1 is phosphorylated by TBK1, but this phosphorylation is not required for downregulation of TBK1 protein. Transfection assays indicate that FTRCA1 is likely an E3 ligase with the requirement of RING finger domain, and deletion of N-terminal RING domain or mutation of seven conservative sites abolishes the negative regulatory function of FTRCA1. Collectively, these results illuminate a novel finTRIM-mediated innate immune modulatory pathway, thus providing insights into species-specific regulation of fish IFN response.


Assuntos
Autofagossomos/imunologia , Proteínas de Peixes/imunologia , Carpa Dourada/imunologia , Interferons/imunologia , Lisossomos/imunologia , Proteínas Serina-Treonina Quinases/imunologia , Proteólise , Proteínas com Motivo Tripartido/imunologia , Animais , Proteínas de Peixes/genética , Carpa Dourada/genética , Células HEK293 , Humanos , Interferons/genética , Lisossomos/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas com Motivo Tripartido/genética
16.
Fish Shellfish Immunol ; 88: 391-402, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30853655

RESUMO

Tripartite motif (TRIM) proteins have been demonstrated to exhibit critical functions in multiple cellular processes, including development, carcinogenesis, and programmed cell death, and are also widely recognized to be important antiviral restriction factors or modulators of immune and inflammatory signaling pathways. However, in teleosts, additional TRIM members have been identified and their functions remain largely unknown. Here, a novel finTRIM gene from orange spotted grouper (EcfinTRIM82) was cloned and characterized. Sequence analysis indicated that EcfinTRIM82 encoded a 575 amino acid peptide which shared 94% and 82% identity with Asian sea bass (Lates calcarifer), and zebrafish (Danio rerio) finTRIM82, respectively. EcfinTRIM82 contained three conserved domains, including a RING, B-Box, and SPRY domain. Using fluorescence microscopy, we found that green fluorescence aggregates were observed in the cytoplasm of EcfinTRIM82-EGFP transfected grouper spleen (GS) cells. As the infection proceeded, EcfinTRIM82 transcription was significantly upregulated in Singapore grouper iridovirus (SGIV) or red-spotted grouper nervous necrosis virus (RGNNV) infected GS cells. This suggests that EcfinTRIM82 might be involved in fish virus infection. The in vitro overexpression of EcfinTRIM82 in GS cells significantly enhanced the replication of SGIV and RGNNV, evidenced by increased expression of viral genes, including the SGIV major capsid protein (MCP), VP19, ICP-18, RGNNV coat protein (CP), and RNA-dependent RNA polymerase (RdRp). Furthermore, the ectopic expression of EcfinTRIM82 significantly decreased the expression of interferon (IFN)-related signaling molecules, including interferon regulatory factor 3 (IRF3), IRF7, interferon stimulated gene 15 (ISG15), ISG56, IFP35, and myxovirus resistance gene (MXI), suggesting that EcfinTRIM82 regulated viral replication via the negative regulation of the host IFN response. In addition, EcfinTRIM82 overexpression substantially decreased the level of proinflammatory cytokine transcription. Furthermore, the ectopic expression of EcfinTRIM82 significantly weakened the melanoma differentiation-associated protein 5 (MDA5), mediator of IRF3 activation (MITA) and mitochondrial antiviral-signaling (MAVS) protein-induced IFN response by detecting the transcription of interferon related cytokines and the promoter activity of IFN. Together, our results demonstrate that finTRIM82 negatively regulates the innate antiviral immune response against grouper virus infection.


Assuntos
Bass/imunologia , Infecções por Vírus de DNA/veterinária , Proteínas de Peixes/imunologia , Imunidade Inata , Interferons/imunologia , Proteínas com Motivo Tripartido/imunologia , Animais , Bass/virologia , Clonagem Molecular , Infecções por Vírus de DNA/imunologia , DNA Complementar , Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Regulação da Expressão Gênica , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/imunologia , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/imunologia , Iridovirus/imunologia , Filogenia , RNA Mensageiro , Alinhamento de Sequência , Análise de Sequência de DNA , Baço/citologia , Baço/virologia , Proteínas com Motivo Tripartido/genética , Peixe-Zebra/imunologia
17.
Viruses ; 11(3)2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30871003

RESUMO

The antiviral activity of type I interferons (IFNs) is primarily mediated by interferon-stimulated genes (ISGs). Induction of ISG transcription is achieved when type I IFNs bind to their cognate receptor and activate the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) signaling pathways. Recently it has become clear that a number of viruses are capable of directly upregulating a subset of ISGs in the absence of type I IFN production. Using cells engineered to block either the response to, or production of type I IFN, the regulation of IFN-independent ISGs was examined in the context of human cytomegalovirus (HCMV) infection. Several ISGs, including IFIT1, IFIT2, IFIT3, Mx1, Mx2, CXCL10 and ISG15 were found to be upregulated transcriptionally following HCMV infection independently of type I IFN-initiated JAK-STAT signaling, but dependent on intact IRF3 signaling. ISG15 protein regulation mirrored that of its transcript with IFNß neutralization failing to completely inhibit ISG15 expression post HCMV infection. In addition, no detectable ISG15 protein expression was observed following HCMV infection in IRF3 knockdown CRISPR/Cas-9 clones indicating that IFN-independent control of ISG expression during HCMV infection of human fibroblasts is absolutely dependent on IRF3 expression.


Assuntos
Citomegalovirus/imunologia , Imunidade Inata , Fator Regulador 3 de Interferon/genética , Interferons/imunologia , Transdução de Sinais , Células Cultivadas , Citocinas/genética , Fibroblastos/virologia , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Fator Regulador 3 de Interferon/imunologia , Interferon beta/genética , Reação em Cadeia da Polimerase em Tempo Real , Ubiquitinas/genética , Regulação para Cima
18.
Viruses ; 11(2)2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30791481

RESUMO

Myeloid cells represent a diverse range of innate leukocytes that are crucial for mounting successful immune responses against viruses. These cells are responsible for detecting pathogen-associated molecular patterns, thereby initiating a signaling cascade that results in the production of cytokines such as interferons to mitigate infections. The aim of this review is to outline recent advances in our knowledge of the roles that neutrophils and inflammatory monocytes play in initiating and coordinating host responses against viral infections. A focus is placed on myeloid cell development, trafficking and antiviral mechanisms. Although known for promoting inflammation, there is a growing body of literature which demonstrates that myeloid cells can also play critical regulatory or immunosuppressive roles, especially following the elimination of viruses. Additionally, the ability of myeloid cells to control other innate and adaptive leukocytes during viral infections situates these cells as key, yet under-appreciated mediators of pathogenic inflammation that can sometimes trigger cytokine storms. The information presented here should assist researchers in integrating myeloid cell biology into the design of novel and more effective virus-targeted therapies.


Assuntos
Interações Hospedeiro-Patógeno/imunologia , Inflamação , Células Mieloides/imunologia , Viroses/imunologia , Vírus/imunologia , Animais , Citocinas , Humanos , Imunidade Inata , Interferons/imunologia , Camundongos , Monócitos/imunologia , Células Mieloides/virologia , Neutrófilos/imunologia , Transdução de Sinais
19.
J Immunol ; 202(7): 2121-2130, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30745462

RESUMO

Systemic lupus erythematosus (SLE) is a complex autoimmune disease in which 70% of patients experience disfiguring skin inflammation (grouped under the rubric of cutaneous lupus erythematosus [CLE]). There are limited treatment options for SLE and no Food and Drug Administration-approved therapies for CLE. Studies have revealed that IFNs are important mediators for SLE and CLE, but the mechanisms by which IFNs lead to disease are still poorly understood. We aimed to investigate how IFN responses in SLE keratinocytes contribute to development of CLE. A cohort of 72 RNA sequencing samples from 14 individuals (seven SLE and seven healthy controls) were analyzed to study the transcriptomic effects of type I and type II IFNs on SLE versus control keratinocytes. In-depth analysis of the IFN responses was conducted. Bioinformatics and functional assays were conducted to provide implications for the change of IFN response. A significant hypersensitive response to IFNs was identified in lupus keratinocytes, including genes (IFIH1, STAT1, and IRF7) encompassed in SLE susceptibility loci. Binding sites for the transcription factor PITX1 were enriched in genes that exhibit IFN-sensitive responses. PITX1 expression was increased in CLE lesions based on immunohistochemistry, and by using small interfering RNA knockdown, we illustrated that PITX1 was required for upregulation of IFN-regulated genes in vitro. SLE patients exhibit increased IFN signatures in their skin secondary to increased production and a robust, skewed IFN response that is regulated by PITX1. Targeting these exaggerated pathways may prove to be beneficial to prevent and treat hyperinflammatory responses in SLE skin.


Assuntos
Regulação da Expressão Gênica/imunologia , Interferons/imunologia , Queratinócitos/imunologia , Lúpus Eritematoso Cutâneo/imunologia , Fatores de Transcrição Box Pareados/imunologia , Adulto , Feminino , Humanos , Masculino
20.
RNA ; 25(5): 539-556, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30770398

RESUMO

The interferon-inducible protein kinase R (PKR) is a key component of host innate immunity that restricts viral replication and propagation. As one of the four eIF2α kinases that sense diverse stresses and direct the integrated stress response (ISR) crucial for cell survival and proliferation, PKR's versatile roles extend well beyond antiviral defense. Targeted by numerous host and viral regulators made of RNA and proteins, PKR is subject to multiple layers of endogenous control and external manipulation, driving its rapid evolution. These versatile regulators include not only the canonical double-stranded RNA (dsRNA) that activates the kinase activity of PKR, but also highly structured viral, host, and artificial RNAs that exert a full spectrum of effects. In this review, we discuss our deepening understanding of the allosteric mechanism that connects the regulatory and effector domains of PKR, with an emphasis on diverse structured RNA regulators in comparison to their protein counterparts. Through this analysis, we conclude that much of the mechanistic details that underlie this RNA-regulated kinase await structural and functional elucidation, upon which we can then describe a "PKR code," a set of structural and chemical features of RNA that are both descriptive and predictive for their effects on PKR.


Assuntos
Interações Hospedeiro-Patógeno/genética , RNA de Cadeia Dupla/genética , RNA não Traduzido/genética , Viroses/genética , eIF-2 Quinase/genética , Regulação Alostérica , Animais , Sequência de Bases , Sítios de Ligação , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata , Interferons/genética , Interferons/imunologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/imunologia , RNA não Traduzido/química , RNA não Traduzido/imunologia , Viroses/imunologia , Viroses/virologia , Replicação Viral , eIF-2 Quinase/química , eIF-2 Quinase/imunologia
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