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1.
Science ; 371(6526)2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33446526

RESUMO

Human monogenic disorders have revealed the critical contribution of type 17 responses in mucosal fungal surveillance. We unexpectedly found that in certain settings, enhanced type 1 immunity rather than defective type 17 responses can promote mucosal fungal infection susceptibility. Notably, in mice and humans with AIRE deficiency, an autoimmune disease characterized by selective susceptibility to mucosal but not systemic fungal infection, mucosal type 17 responses are intact while type 1 responses are exacerbated. These responses promote aberrant interferon-γ (IFN-γ)- and signal transducer and activator of transcription 1 (STAT1)-dependent epithelial barrier defects as well as mucosal fungal infection susceptibility. Concordantly, genetic and pharmacologic inhibition of IFN-γ or Janus kinase (JAK)-STAT signaling ameliorates mucosal fungal disease. Thus, we identify aberrant T cell-dependent, type 1 mucosal inflammation as a critical tissue-specific pathogenic mechanism that promotes mucosal fungal infection susceptibility in mice and humans.


Assuntos
Candida albicans/imunologia , Candidíase Mucocutânea Crônica/genética , Candidíase Mucocutânea Crônica/imunologia , Imunidade nas Mucosas/imunologia , Poliendocrinopatias Autoimunes/genética , Poliendocrinopatias Autoimunes/imunologia , Adolescente , Adulto , Idoso , Animais , Modelos Animais de Doenças , Feminino , Humanos , Imunidade nas Mucosas/genética , Vigilância Imunológica/genética , Vigilância Imunológica/imunologia , Interferon gama/genética , Interleucinas/genética , Janus Quinases/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Mucosa Bucal/imunologia , Mucosa Bucal/patologia , Receptores de Interleucina-17/genética , Fator de Transcrição STAT1/genética , Linfócitos T/imunologia , Adulto Jovem
2.
J Cancer Res Clin Oncol ; 147(3): 725-737, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33387041

RESUMO

PURPOSE: Crizotinib is the first-line small molecule tyrosine kinase inhibitor for ALK-positive non-small cell lung cancer. In this study, a retrospective pharmacogenomics investigation was conducted to explore the relationship between genes related to RTK downstream signaling pathways and crizotinib-induced hepatic toxicity in ALK-positive NSCLC patients. METHODS: The variable importance analysis of random forest algorithm was applied to identify the significant features which contribute to the crizotinib sensitivity in Cancer Cell Line Encyclopedia (CCLE) database. The KEGG and reactome pathway enrichment analysis were conducted with EnrichR. The differential expression genes were identified with R package DESeq2 in CCLE liver derived cell lines between crizotinib sensitive and resistant groups. From 2012 to 2015, 42 NSCLC patients were enrolled in this study. 90 polymorphisms were genotyped using the Sequenom Massarray system. Sequencing of HGFR (c-Met) genes was carried out on the Ion Torrent Proton. RESULTS: In total, 66.7% NSCLC patients suffered from crizotinib-induced liver toxicity and 11.9% progressed to severe hepatic toxicity. The features with the top importance from classification and regression random forest model were enriched in RTK downstream signaling pathways (JAK/STAT, RAS/RAF/MAPK, PI3K/AKT pathways) and immune system-related pathways. Collagen family genes (COL1A1, COL1A2, COL6A1, COL5A1) and other extracellular matrix protein (TNC, TAGLN, TENM2, EDIL3, VCAN, CNN1, SH3BP4, TAGLN), which were closely related to MAPK-ERK signaling pathways, were significantly enriched in crizotinib resistant cell lines. In multiple logistic regression, STAT1 rs10208033 (T > C) was significantly associated with crizotinib-induced liver toxicity (OR = 6.733, 95% CI 1.406-32.24, P = 0.017). Compared with non-CC, OR is 5.5 (95% CI 1.219-24.81, P = 0.027) for STAT1 rs10208033 CC genotype to develop crizotinib-induced liver toxicity. Further cell viability test in human fetal hepatocyte line, L-02, reveals that the STAT1 inhibitor might protect hepatocyte cells from the toxicity caused by crizotinib. CONCLUSION: Polymorphism of rs10208033 is a potential biomarker for predicting crizotinib-induced hepatotoxicity. These results suggest that STAT1 plays an important role in crizotinib-induced hepatotoxicity. Further studies are needed to confirm our finding and understand the underlying mechanisms.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Doença Hepática Induzida por Substâncias e Drogas/genética , Crizotinibe/efeitos adversos , Neoplasias Pulmonares/genética , Fator de Transcrição STAT1/genética , Adulto , Idoso , Quinase do Linfoma Anaplásico/antagonistas & inibidores , Quinase do Linfoma Anaplásico/metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Crizotinibe/farmacologia , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Masculino , Pessoa de Meia-Idade , Polimorfismo Genético , Inibidores de Proteínas Quinases/efeitos adversos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/genética , Estudos Retrospectivos , Transdução de Sinais
5.
Phytomedicine ; 80: 153371, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33070080

RESUMO

BACKGROUND: Apigenin is one of the most abundant dietary flavonoids that possesses multiple bio-functions. PURPOSE: This study was designed to determine the influence of apigenin on gene expressions, cancer cells, as well as STAT1/COX-2/iNOS pathway mediated inflammation and tumorigenesis in HEK293-STAT1 cells. Furthermore, the cytotoxic activity toward multiple myeloma (MM) cell lines was investigated. METHODS: Bioinformatic analyses were used to predict the sensitivity and resistance of tumor cells toward apigenin and to determine cellular pathways influenced by this compound. The cytotoxic and ferroptotic activity of apigenin was examined by the resazurin reduction assay. Additionally, we evaluated apoptosis, and cell cycle distribution, induction of reactive oxygen species (ROS) and loss of integrity of mitochondrial membrane (MMP) by using the flow cytometry analysis. DAPI staining was used to detect characteristic apoptotic features. Furthermore, we verified its anti-inflammatory and additional mechanism of cell death by western blotting. RESULTS: COMPARE and hierarchical cluster analyses exhibited that 29 of 55 tumor cell lines were sensitive against apigenin (p < 0.001). The Ingenuity Pathway Analysis data showed that important bio-functions affected by apigenin were: gene expression, cancer, hematological system development and function, inflammatory response, and cell cycle. The STAT1 transcription factor was chosen as target protein on the basis of gene promoter binding motif analyses. Apigenin blocked cell proliferation of wild-type HEK293 and STAT1 reporter cells (HEK293-STAT1), promoted STAT1 suppression and subsequent COX-2 and iNOS inhibition. Apigenin also exhibited synergistic activity in combination with doxorubicin toward HEK293-STAT1 cells. Apigenin exerted excellent growth-inhibitory activity against MM cells in a concentration-dependent manner with the greatest activity toward NCI-H929 (IC50 value: 10.73 ± 3.21 µM). Apigenin induced apoptosis, cell cycle arrest, ferroptosis and autophagy in NCI-H929 cells. CONCLUSION: Apigenin may be a suitable candidate for MM treatment. The inhibition of the STAT1/COX-2/iNOS signaling pathway by apigenin is an important mechanism not only in the suppression of inflammation but also in induction of apoptosis.


Assuntos
Apigenina/farmacologia , Ciclo-Oxigenase 2/metabolismo , Mieloma Múltiplo/tratamento farmacológico , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Fator de Transcrição STAT1/genética , Antineoplásicos Fitogênicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apigenina/administração & dosagem , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Biologia Computacional/métodos , Relação Dose-Resposta a Droga , Doxorrubicina/administração & dosagem , Células HEK293 , Humanos , Mieloma Múltiplo/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição STAT1/metabolismo
6.
Signal Transduct Target Ther ; 5(1): 221, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-33024073
8.
PLoS Pathog ; 16(9): e1008767, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32903273

RESUMO

Many viruses target signal transducer and activator of transcription (STAT) 1 to antagonise antiviral interferon signalling, but targeting of STAT3, a pleiotropic molecule that mediates signalling by diverse cytokines, is poorly understood. Here, using lyssavirus infection, quantitative live cell imaging, innate immune signalling and protein interaction assays, and complementation/depletion of STAT expression, we show that STAT3 antagonism is conserved among P-proteins of diverse pathogenic lyssaviruses and correlates with pathogenesis. Importantly, P-protein targeting of STAT3 involves a highly selective mechanism whereby P-protein antagonises cytokine-activated STAT3-STAT1 heterodimers, but not STAT3 homodimers. RT-qPCR and reporter gene assays indicate that this results in specific modulation of interleukin-6-dependent pathways, effecting differential antagonism of target genes. These data provide novel insights into mechanisms by which viruses can modulate cellular function to support infection through discriminatory targeting of immune signalling complexes. The findings also highlight the potential application of selective interferon-antagonists as tools to delineate signalling by particular STAT complexes, significant not only to pathogen-host interactions but also cell physiology, development and cancer.


Assuntos
Citocinas/metabolismo , Regulação da Expressão Gênica , Lyssavirus/imunologia , Infecções por Rhabdoviridae/imunologia , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/metabolismo , Proteínas Virais/metabolismo , Células HEK293 , Células HeLa , Humanos , Interleucina-6/metabolismo , Infecções por Rhabdoviridae/metabolismo , Infecções por Rhabdoviridae/virologia , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT3/genética , Transativadores , Proteínas Virais/genética
9.
PLoS One ; 15(9): e0239952, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32991625

RESUMO

Signal transducer and activator of transcription 1 (STAT1) is known to be an important player in inflammatory responses. STAT1 as a transcription factor regulates the expression of multiple proinflammatory genes. Inflammatory response is one of the common effects of ototoxicity. Our group reported that hair cells of STAT1 knockout (STAT1-KO) mice are less sensitive to ototoxic agents in-vitro. The effect of inflammatory responses in STAT1-KO mice has primarily been studied challenging them with several pathogens and analyzing different organs of those mice. However, the effect of STAT1 ablation in the mouse inner ear has not been reported. Therefore, we evaluated the cochlear function of wild type and STAT1-KO mice via auditory brain stem response (ABR) and performed histopathologic analysis of their temporal bones. We found ABR responses were affected in STAT1-KO mice with cases of bilateral and unilateral hearing impairment. Histopathologic examination of the middle and inner ears showed bilateral and unilateral otitis media. Otitis media was characterized by effusion of middle and inner ear that varied between the mice in volume and inflammatory cell content. In addition, the thickness of the middle ear mucosae in STAT1-KO mice were more pronounced than those in wild type mice. The degree of middle and inner ear inflammation correlated with ABR threshold elevation in STAT1-KO mice. It appears that a number of mice with inflammation underwent spontaneous resolution. The ABR thresholds were variable and showed a tendency to increase in homozygous and heterozygous STAT1-KO mice. These findings suggest that STAT1 ablation confers an increased susceptibility to otitis media leading to hearing impairment. Thus, the study supports the new role of STAT1 as otitis media predisposition gene.


Assuntos
Otite Média/genética , Fator de Transcrição STAT1/genética , Animais , Cóclea/patologia , Cóclea/fisiopatologia , Orelha Média/patologia , Orelha Média/fisiopatologia , Potenciais Evocados Auditivos do Tronco Encefálico , Camundongos , Camundongos Endogâmicos C57BL , Fator de Transcrição STAT1/deficiência
10.
Medicine (Baltimore) ; 99(37): e22092, 2020 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-32925750

RESUMO

Pancreatic cancer (PaCa) is one of the most fatal cancers in the world. Although great efforts have made to explore the mechanisms of PaCa oncogenesis, the prognosis of PaCa patients is still unsatisfactory. Thus, it is imperative to further understand the potential carcinogenesis of PaCa and reliable prognostic models.The gene expression profile and clinical information of GSE21501 were downloaded from the Gene Expression Omnibus (GEO) database. Weighted gene co-expression network analysis (WGCNA) was applied to explore the potent genes associated with the overall survival (OS) events of PaCa patients. Cox regression model was applied to selecting prognostic genes and establish prognostic model. The prognostic values of six-gene signature were validated in TCGA-PAAD cohort.According to the WGCNA analysis, a total of 19 modules were identified and 115 hub genes in the mostly associated module were reserved for next analysis. According to the univariate and multivariate Cox regression analysis, we established a six-gene signature (FTSJ3, STAT1, STX2, CDX2, RASSF4, MACF1) which could effectively evaluate the overall survival (OS) of PaCa patients. In validated patients' cohorts, the six-gene signature exhibited excellent prognostic value in TCGA-PAAD cohort as well.We developed a six-gene signature to exactly predict OS of PaCa patients and provide a novel personalized strategy for evaluating prognosis. The findings may be contributed to medical customization and therapeutic decision in clinical practice.


Assuntos
Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/mortalidade , Fator de Transcrição CDX2/genética , Perfilação da Expressão Gênica , Humanos , Metiltransferases/genética , Proteínas dos Microfilamentos/genética , Modelos Estatísticos , Neoplasias Pancreáticas/patologia , Valor Preditivo dos Testes , Prognóstico , Modelos de Riscos Proporcionais , Fator de Transcrição STAT1/genética , Sintaxina 1/genética , Proteínas Supressoras de Tumor/genética
11.
Ecotoxicol Environ Saf ; 205: 111154, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32810643

RESUMO

The study focused on the toxicological effect of Di-n-butyl phthalate (DBP) on the expression of Phosphorylated signal transducer and activator of transcription 1 (pSTAT1) -regulated Forkhead box protein M1 (FoxM1), which might provide a new understanding of gestational diabetes mellitus (GDM) development and a potential target for treatment. Streptozotocin (STZ) (40 mg/kg) was introduced in maternal rats by intraperitoneal injection on gestation day 0 (GD 0) in the STZ and STZ + DBP groups. DBP was introduced in maternal rats by oral feeding in the STZ + DBP group over the following 3 days (750 mg/kg/day). The changes in fasting blood glucose level in rats were detected on GD 1 and GD 5. The insulin levels in maternal rats and PIBCs were measured on GD 18. The Oral Glucose Tolerance Test (OGTT) test was performed on GD 18 to check the stability of the GDM model. The primary islet ß cells (PIBCs) were established for in vitro experiments. We examined the FoxM1 and pSTAT1 expression in pancreas by immunohistochemistry. Real-time PCR and Western blot were used to detect the pSTAR1 and FoxM1 protein and mRNA gene expression levels in PIBCs. Cell Counting Kit-8 (CCK-8) and flow cytometric analysis was used to test the viability and apoptosis of cells. The results showed that the STZ + DBP group had higher glucose and lower insulin secretion levels than the other groups by both fasting test and OGTT. FoxM1 was significantly suppressed while pSTAT1 was highly expressed after DBP exposure. FoxM1 could be regulated by pSTAT1. DBP can influence the progression of GDM through its toxicological effect, which significantly increases the expression of pSTAT1 and suppresses FoxM1, causing a decline in ß cell viability.


Assuntos
Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Gestacional/induzido quimicamente , Dibutilftalato/toxicidade , Disruptores Endócrinos/toxicidade , Proteína Forkhead Box M1/metabolismo , Exposição Materna/efeitos adversos , Fator de Transcrição STAT1/metabolismo , Animais , Células Cultivadas , Diabetes Mellitus Experimental/metabolismo , Diabetes Gestacional/metabolismo , Feminino , Proteína Forkhead Box M1/genética , Expressão Gênica/efeitos dos fármacos , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Masculino , Fosforilação , Gravidez , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Fator de Transcrição STAT1/genética , Transdução de Sinais
12.
J Virol ; 94(18)2020 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-32759316

RESUMO

An entirely plasmid-based reverse genetics (RG) system was recently developed for rotavirus (RV), opening new avenues for in-depth molecular dissection of RV biology, immunology, and pathogenesis. Several improvements to further optimize the RG efficiency have now been described. However, only a small number of individual RV strains have been recovered to date. None of the current methods have supported the recovery of murine RV, impeding the study of RV replication and pathogenesis in an in vivo suckling mouse model. Here, we describe useful modifications to the RG system that significantly improve rescue efficiency of multiple RV strains. In addition to the 11 group A RV segment-specific (+)RNAs [(+)ssRNAs], a chimeric plasmid was transfected, from which the capping enzyme NP868R of African swine fever virus (ASFV) and the T7 RNA polymerase were expressed. Second, a genetically modified MA104 cell line was used in which several components of the innate immunity were degraded. Using this RG system, we successfully recovered the simian RV RRV strain, the human RV CDC-9 strain, a reassortant between murine RV D6/2 and simian RV SA11 strains, and several reassortants and reporter RVs. All these recombinant RVs were rescued at a high efficiency (≥80% success rate) and could not be reliably rescued using several recently published RG strategies (<20%). This improved system represents an important tool and great potential for the rescue of other hard-to-recover RV strains such as low-replicating attenuated vaccine candidates or low-cell culture passage clinical isolates from humans or animals.IMPORTANCE Group A rotavirus (RV) remains as the single most important cause of severe acute gastroenteritis among infants and young children worldwide. An entirely plasmid-based reverse genetics (RG) system was recently developed, opening new ways for in-depth molecular study of RV. Despite several improvements to further optimize the RG efficiency, it has been reported that current strategies do not enable the rescue of all cultivatable RV strains. Here, we described a helpful modification to the current strategies and established a tractable RG system for the rescue of the simian RRV strain, the human CDC-9 strain, and a murine-like RV strain, which is suitable for both in vitro and in vivo studies. This improved RV reverse genetics system will facilitate study of RV biology in both in vitro and in vivo systems that will facilitate the improved design of RV vaccines, better antiviral therapies, and expression vectors.


Assuntos
Regulação Viral da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Vírus Reordenados/genética , Genética Reversa/métodos , Rotavirus/genética , Proteínas Virais/genética , Vírus da Febre Suína Africana/genética , Vírus da Febre Suína Africana/imunologia , Animais , Chlorocebus aethiops , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/imunologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata , Fatores Reguladores de Interferon/deficiência , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/imunologia , Camundongos , Nucleotidiltransferases/genética , Nucleotidiltransferases/imunologia , Plasmídeos/química , Plasmídeos/metabolismo , Capuzes de RNA , Vírus Reordenados/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Rotavirus/imunologia , Fator de Transcrição STAT1/deficiência , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/imunologia , Transfecção , Células Vero , Proteínas Virais/imunologia , Replicação Viral
13.
Cell ; 182(3): 734-743.e5, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32643603

RESUMO

COVID-19, caused by SARS-CoV-2, is a virulent pneumonia, with >4,000,000 confirmed cases worldwide and >290,000 deaths as of May 15, 2020. It is critical that vaccines and therapeutics be developed very rapidly. Mice, the ideal animal for assessing such interventions, are resistant to SARS-CoV-2. Here, we overcome this difficulty by exogenous delivery of human ACE2 with a replication-deficient adenovirus (Ad5-hACE2). Ad5-hACE2-sensitized mice developed pneumonia characterized by weight loss, severe pulmonary pathology, and high-titer virus replication in lungs. Type I interferon, T cells, and, most importantly, signal transducer and activator of transcription 1 (STAT1) are critical for virus clearance and disease resolution in these mice. Ad5-hACE2-transduced mice enabled rapid assessments of a vaccine candidate, of human convalescent plasma, and of two antiviral therapies (poly I:C and remdesivir). In summary, we describe a murine model of broad and immediate utility to investigate COVID-19 pathogenesis and to evaluate new therapies and vaccines.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/patologia , Infecções por Coronavirus/prevenção & controle , Modelos Animais de Doenças , Pandemias/prevenção & controle , Pneumonia Viral/patologia , Pneumonia Viral/prevenção & controle , Vacinação , Animais , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Humanos , Interferon gama/genética , Interferon gama/metabolismo , Pulmão/patologia , Pulmão/virologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/virologia , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/metabolismo , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Organismos Livres de Patógenos Específicos , Transdução Genética , Células Vero , Carga Viral , Replicação Viral
14.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 28(3): 821-827, 2020 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-32552942

RESUMO

OBJECTIVE: To analyze the relationships between expression levels of serum microRNA-146a, STAT1 protein and clinical characteristics in children with acute lymphoblastic leukemia (ALL). METHODS: A total of 102 children diagnosed as ALL in our hospital from June 2014 to June 2016 were enrolled, and were compared by into groups according to clinical characteristics including sex, age, lymphocyte type, disease risk, chemotherapy stage and gene mutation. Fifty healthy children were chosen as control group. The relative expression of microRNA-146a and STAT1 gene was detected by real-time RT-PCR and the relative level of STAT1 protein was detected by Western blot. The difference of microRNA-146a and STAT1 protein levels between clinical factors and laboratory indexs were compared. Followed-up for 3 years, The difference of overall survival (OS) rates between ALL children with different microRNA-146a and STAT1 protein were compared. RESULTS: The levels of microRNA-146a, STAT1 mRNA and protein in ALL children were significantly higher than those in control group (P<0.05), but there were no significantly differences in sex, age and lymphocyte type grouping in ALL children (P>0.05). There were significantly differences in different disease risk, chemotherapy stage and gene mutation groups in ALL children (P<0.05). Followed-up for 3 years, the OS rate of ALL children with high microRNA-146a and STAT1 protein levels were better than those with low microRNA-146a and STAT1 protein levels (P<0.05). CONCLUSION: The up-regulation of microRNA-146a and STAT1 protein may be involved in occurrence and development of ALL, which closely relates to clinical characteristics in ALL children, such as disease risk, chemotherapy stage and gene mutation.


Assuntos
MicroRNAs/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras , Fator de Transcrição STAT1/genética , Criança , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , RNA Mensageiro , Regulação para Cima
15.
J Virol ; 94(17)2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32581091

RESUMO

Measles virus (MeV) is a highly immunotropic and contagious pathogen that can even diminish preexisting antibodies and remains a major cause of childhood morbidity and mortality worldwide despite the availability of effective vaccines. MeV is one of the most extensively studied viruses with respect to the mechanisms of JAK-STAT antagonism. Of the three proteins translated from the MeV P gene, P and V are essential for inactivation of this pathway. However, the lack of data from direct analyses of the underlying interactions means that the detailed molecular mechanism of antagonism remains unresolved. Here, we prepared recombinant MeV V protein, which is responsible for human JAK-STAT antagonism, and a panel of variants, enabling the biophysical characterization of V protein, including direct V/STAT1 and V/STAT2 interaction assays. Unambiguous direct interactions between the host and viral factors, in the absence of other factors such as Jak1 or Tyk2, were observed, and the dissociation constants were quantified for the first time. Our data indicate that interactions between the C-terminal region of V and STAT2 is 1 order of magnitude stronger than that of the N-terminal region of V and STAT1. We also clarified that these interactions are completely independent of each other. Moreover, results of size exclusion chromatography demonstrated that addition of MeV-V displaces STAT2-core, a rigid region of STAT2 lacking the N- and C-terminal domains, from preformed complexes of STAT2-core/IRF-associated domain (IRF9). These results provide a novel model whereby MeV-V can not only inhibit the STAT2/IRF9 interaction but also disrupt preassembled interferon-stimulated gene factor 3.IMPORTANCE To evade host immunity, many pathogenic viruses inactivate host Janus kinase signal transducer and activator of transcription (STAT) signaling pathways using diverse strategies. Measles virus utilizes P and V proteins to counteract this signaling pathway. Data derived largely from cell-based assays have indicated several amino acid residues of P and V proteins as important. However, biophysical properties of V protein or its direct interaction with STAT molecules using purified proteins have not been studied. We have developed novel molecular tools enabling us to identify a novel molecular mechanism for immune evasion whereby V protein disrupts critical immune complexes, providing a clear strategy by which measles virus can suppress interferon-mediated antiviral gene expression.


Assuntos
Fator Gênico 3 Estimulado por Interferon, Subunidade gama/química , Vírus do Sarampo/metabolismo , Fosfoproteínas/química , Fator de Transcrição STAT2/química , Proteínas Virais/química , Sítios de Ligação , Expressão Gênica , Humanos , Evasão da Resposta Imune , Imunidade Inata , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/genética , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/metabolismo , Janus Quinases/metabolismo , Vírus do Sarampo/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ligação Proteica , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , Fator de Transcrição STAT1/química , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT2/genética , Fator de Transcrição STAT2/metabolismo , Transdução de Sinais , Proteínas Virais/genética , Proteínas Virais/metabolismo , Dedos de Zinco
16.
Anim Sci J ; 91(1): e13378, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32329195

RESUMO

Interferon-tau (IFNT) regulates maternal recognition during early pregnancy in ruminants. The liver can serve as a hematopoietic organ, and it has immune functions. This study hypothesized whether mRNA and proteins of interferon-stimulated genes (ISGs) induced by early pregnancy are upregulated in maternal liver. Therefore, we determined the expression of interferon-stimulated gene 15-kDa protein (ISG15), 2',5'-oligoadenylate synthetase 1 (OAS1), myxovirus resistance protein 1 (MX1), interferon-gamma-inducible protein 10 (IP-10), and signal transducer and activator of transcription 1 (STAT1) in maternal livers during early pregnancy in sheep. Ovine livers were sampled on day 16 of the estrous cycle, and days 13, 16, and 25 of pregnancy, and expression of ISGs was detected by quantitative real-time PCR, Western blot, and immunohistochemistry analysis. Our results showed that there were increases in expression of the mRNA and proteins of ISG15, OAS1, IP-10, STAT1, and MX1 during early pregnancy. STAT1 protein was limited to the hepatocytes, and endothelial cells of proper hepatic arteries and hepatic portal veins. In conclusion, the upregulation of ISG15, OAS1, IP-10, STAT1, and MX1 proteins may be implicated in maternal hepatic immune adjustment and other functions during early pregnancy in sheep.


Assuntos
2',5'-Oligoadenilato Sintetase/genética , 2',5'-Oligoadenilato Sintetase/metabolismo , Quimiocina CXCL10/genética , Quimiocina CXCL10/metabolismo , Citocinas/genética , Citocinas/metabolismo , Expressão Gênica , Fígado/metabolismo , Proteínas de Resistência a Myxovirus/genética , Proteínas de Resistência a Myxovirus/metabolismo , Prenhez/metabolismo , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Ovinos/genética , Ovinos/fisiologia , Ubiquitinas/genética , Ubiquitinas/metabolismo , Animais , Ciclo Estral/genética , Ciclo Estral/metabolismo , Feminino , Fígado/imunologia , Gravidez , Prenhez/imunologia , Ovinos/imunologia , Regulação para Cima
17.
Oncogene ; 39(22): 4344-4357, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32335582

RESUMO

We explore a novel strategy of activating immune signaling through increased micronuclei formation utilizing a cell cycle checkpoint inhibitor to drive cell cycle progression following ionizing radiation. The Chk1/2 inhibitor AZD7762 is used to abrogate radiation therapy (RT)-induced G2/M cell cycle arrest in multiple cell lines and, we find that this therapeutic combination promotes increased micronuclei formation in vitro and subsequently drives increased type I interferon signaling and cytotoxic T-cell activation. In vivo studies using B16-F10 melanoma cancer cells implanted in C57/BL6 mice demonstrate improved rates of tumor control at the abscopal (unirradiated) site, located outside of the radiation field, only in the AZD7762 + RT group, with a corresponding reduction in mean tumor volume, increase in the CD8 T-cell population, and immune activated gene signaling. Our results demonstrate that targeted inhibition of cell cycle checkpoint activation following ionizing radiation drives increased production of immunogenic micronuclei, leading to systemic tumor response with potential future clinical benefit.


Assuntos
Núcleo Celular/efeitos dos fármacos , Núcleo Celular/efeitos da radiação , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Quinase do Ponto de Checagem 2/antagonistas & inibidores , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos da radiação , Melanoma Experimental/imunologia , Proteínas de Neoplasias/antagonistas & inibidores , Tiofenos/farmacologia , Ureia/análogos & derivados , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Interferon beta/biossíntese , Interferon beta/genética , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/patologia , Melanoma Experimental/radioterapia , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Testes para Micronúcleos , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Fator de Transcrição STAT1/biossíntese , Fator de Transcrição STAT1/genética , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/efeitos da radiação , Ureia/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
18.
J Virol ; 94(12)2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32295905

RESUMO

We demonstrate that female C57BL/6J mice are susceptible to a transient lower genital tract infection with MmuPV1 mouse papillomavirus and display focal histopathological abnormalities resembling those of human papillomavirus (HPV) infection. We took advantage of strains of genetically deficient mice to study in vivo the role of innate immune signaling in the control of papillomavirus. At 4 months, we sacrificed MmuPV1-infected mice and measured viral 757/3139 spliced transcripts by TaqMan reverse transcription-PCR (RT-PCR), localization of infection by RNAscope in situ hybridization, and histopathological abnormities by hematoxylin and eosin (H&E) staining. Among mice deficient in receptors for pathogen-associated molecular patterns, MyD88-/- and STING-/- mice had 1,350 and 80 copies of spliced transcripts/µg RNA, respectively, while no viral expression was detected in MAVS-/- and Ripk2-/- mice. Mice deficient in an adaptor molecule, STAT1-/-, for interferon signaling had 46,000 copies/µg RNA. Among mice with targeted deficiencies in the inflammatory response, interleukin-1 receptor knockout (IL-1R-/-) and caspase-1-/- mice had 350 and 30 copies/µg RNA, respectively. Among mice deficient in chemokine receptors, CCR6-/- mice had 120 copies/µg RNA, while CXCR2-/- and CXCR3-/- mice were negative. RNAscope confirmed focal infection in MyD88-/-, STAT1-/-, and CCR6-/- mice but was negative for other gene-deficient mice. Histological abnormalities were seen only in the latter mice. Our findings and the literature support a working model of innate immunity to papillomaviruses involving the activation of a MyD88-dependent pathway and IL-1 receptor signaling, control of viral replication by interferon-stimulated genes, and clearance of virus-transformed dysplastic cells by the action of the CCR6/CCL20 axis.IMPORTANCE Papillomaviruses infect stratified squamous epithelia, and the viral life cycle is linked to epithelial differentiation. Additionally, changes occur in viral and host gene expression, and immune cells are activated to modulate the infectious process. In vitro studies with keratinocytes cannot fully model the complex viral and host responses and do not reflect the contribution of local and migrating immune cells. We show that female C57BL/6J mice are susceptible to a transient papillomavirus cervicovaginal infection, and mice deficient in select genes involved in innate immune responses are susceptible to persistent infection with variable manifestations of histopathological abnormalities. The results of our studies support a working model of innate immunity to papillomaviruses, and the model provides a framework for more in-depth studies. A better understanding of mechanisms of early viral clearance and the development of approaches to induce clearance will be important for cancer prevention and the treatment of HPV-related diseases.


Assuntos
Interações Hospedeiro-Patógeno/imunologia , Fator 88 de Diferenciação Mieloide/imunologia , Papillomaviridae/imunologia , Infecções por Papillomavirus/imunologia , RNA Mensageiro/imunologia , RNA Viral/imunologia , Receptores Tipo I de Interleucina-1/imunologia , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Processamento Alternativo , Animais , Caspase 1/deficiência , Caspase 1/genética , Caspase 1/imunologia , Colo do Útero/imunologia , Colo do Útero/virologia , Feminino , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Humanos , Imunidade Inata , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/deficiência , Fator 88 de Diferenciação Mieloide/genética , Papillomaviridae/crescimento & desenvolvimento , Papillomaviridae/metabolismo , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/virologia , RNA Mensageiro/genética , RNA Viral/genética , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/deficiência , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/genética , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/imunologia , Receptores CCR6/deficiência , Receptores CCR6/genética , Receptores CCR6/imunologia , Receptores CXCR3/deficiência , Receptores CXCR3/genética , Receptores CXCR3/imunologia , Receptores Tipo I de Interleucina-1/deficiência , Receptores Tipo I de Interleucina-1/genética , Receptores de Interleucina-8B/deficiência , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/imunologia , Fator de Transcrição STAT1/deficiência , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/imunologia , Transdução de Sinais , Vagina/imunologia , Vagina/virologia
19.
Proc Natl Acad Sci U S A ; 117(16): 9022-9031, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32284404

RESUMO

The vast majority of type 1 diabetes (T1D) genetic association signals lie in noncoding regions of the human genome. Many have been predicted to affect the expression and secondary structure of long noncoding RNAs (lncRNAs), but the contribution of these lncRNAs to the pathogenesis of T1D remains to be clarified. Here, we performed a complete functional characterization of a lncRNA that harbors a single nucleotide polymorphism (SNP) associated with T1D, namely, Lnc13 Human pancreatic islets harboring the T1D-associated SNP risk genotype in Lnc13 (rs917997*CC) showed higher STAT1 expression than islets harboring the heterozygous genotype (rs917997*CT). Up-regulation of Lnc13 in pancreatic ß-cells increased activation of the proinflammatory STAT1 pathway, which correlated with increased production of chemokines in an allele-specific manner. In a mirror image, Lnc13 gene disruption in ß-cells partially counteracts polyinosinic-polycytidylic acid (PIC)-induced STAT1 and proinflammatory chemokine expression. Furthermore, we observed that PIC, a viral mimetic, induces Lnc13 translocation from the nucleus to the cytoplasm promoting the interaction of STAT1 mRNA with (poly[rC] binding protein 2) (PCBP2). Interestingly, Lnc13-PCBP2 interaction regulates the stability of the STAT1 mRNA, sustaining inflammation in ß-cells in an allele-specific manner. Our results show that the T1D-associated Lnc13 may contribute to the pathogenesis of T1D by increasing pancreatic ß-cell inflammation. These findings provide information on the molecular mechanisms by which disease-associated SNPs in lncRNAs influence disease pathogenesis and open the door to the development of diagnostic and therapeutic approaches based on lncRNA targeting.


Assuntos
Diabetes Mellitus Tipo 1/genética , Células Secretoras de Insulina/imunologia , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fator de Transcrição STAT1/genética , Regiões 3' não Traduzidas/genética , Sobrevivência Celular/genética , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/virologia , Predisposição Genética para Doença , Células HEK293 , Humanos , Células Secretoras de Insulina/patologia , Células Secretoras de Insulina/virologia , Células Jurkat , Poli I-C/imunologia , Polimorfismo de Nucleotídeo Único , Cultura Primária de Células , Estabilidade de RNA/genética , RNA Mensageiro/metabolismo , RNA Viral/imunologia , Fator de Transcrição STAT1/imunologia , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Regulação para Cima/imunologia
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