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1.
Nat Commun ; 12(1): 723, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33526787

RESUMO

Bone metastatic prostate cancer (PCa) promotes mesenchymal stem cell (MSC) recruitment and their differentiation into osteoblasts. However, the effects of bone-marrow derived MSCs on PCa cells are less explored. Here, we report MSC-derived interleukin-28 (IL-28) triggers prostate cancer cell apoptosis via IL-28 receptor alpha (IL-28Rα)-STAT1 signaling. However, chronic exposure to MSCs drives the selection of prostate cancer cells that are resistant to IL-28-induced apoptosis and therapeutics such as docetaxel. Further, MSC-selected/IL-28-resistant prostate cancer cells grow at accelerated rates in bone. Acquired resistance to apoptosis is PCa cell intrinsic, and is associated with a shift in IL-28Rα signaling via STAT1 to STAT3. Notably, STAT3 ablation or inhibition impairs MSC-selected prostate cancer cell growth and survival. Thus, bone marrow MSCs drive the emergence of therapy-resistant bone metastatic prostate cancer yet this can be disabled by targeting STAT3.


Assuntos
Adenocarcinoma/secundário , Neoplasias Ósseas/secundário , Células-Tronco Mesenquimais/patologia , Neoplasias da Próstata/patologia , Receptores de Interferon/metabolismo , Ácidos Aminossalicílicos/farmacologia , Ácidos Aminossalicílicos/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Benzenossulfonatos/farmacologia , Benzenossulfonatos/uso terapêutico , Neoplasias Ósseas/tratamento farmacológico , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células , Meios de Cultivo Condicionados/metabolismo , Modelos Animais de Doenças , Docetaxel/farmacologia , Docetaxel/uso terapêutico , Humanos , Interferons/genética , Interferons/metabolismo , Masculino , Camundongos Knockout , Osteoblastos/patologia , Cultura Primária de Células , Neoplasias da Próstata/tratamento farmacológico , RNA Interferente Pequeno/metabolismo , Receptores de Interferon/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/metabolismo , Tíbia/patologia
4.
Phytomedicine ; 80: 153340, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33130471

RESUMO

BACKGROUND: Oleanolic acid (OA) is an active compound found in a variety of medicinal herbs and plants. Though OA has been widely attributed with a variety of biological activities, studies focused on its anti-allergic inflammation properties are insufficient. PURPOSE: Given the rapid increase in allergic diseases and the lack of fundamental treatment options, this study aimed to find a safe and effective therapy for allergic disorders. METHODS: We evaluated the inhibitory effect of OA on allergic inflammatory response and the possible mechanisms underlying the effect using phorbol-12-myristate 13-acetate plus calcium ionophore A23187 (PMACI)-stimulated human mast cell (HMC)-1, and a mouse model of compound 48/80-induced anaphylactic shock. RESULTS: OA suppressed pro-inflammatory cytokine expressions in PMACI-induced HMC-1 cells by inhibiting activation of the Akt, p38 mitogen-activated protein kinase (MAPK), nuclear factor-κB (NF-κB), and signal transducer and activator of transcription (STAT) 1 signaling pathways. Moreover, OA showed a protective effect against compound 48/80-induced anaphylactic shock through inhibition of histamine release and immunoglobulin E level via regulation of NF-κB and STAT1 activation. CONCLUSION: The results showed that OA suppressed mast cell-mediated allergic response by transcriptional regulation. We suggest that OA has potential effect against allergic inflammatory disorders, including anaphylaxis, and might be a useful therapeutic agent for allergic disease.


Assuntos
Anafilaxia/prevenção & controle , Antialérgicos/farmacologia , Mastócitos/efeitos dos fármacos , Ácido Oleanólico/farmacologia , Anafilaxia/induzido quimicamente , Animais , Calcimicina/toxicidade , Linhagem Celular , Citocinas/metabolismo , Liberação de Histamina/efeitos dos fármacos , Humanos , Inflamação/tratamento farmacológico , Mediadores da Inflamação/metabolismo , Masculino , Mastócitos/metabolismo , Camundongos Endogâmicos ICR , NF-kappa B/metabolismo , Ésteres de Forbol/toxicidade , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição STAT1/metabolismo , p-Metoxi-N-metilfenetilamina/toxicidade , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
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.
Biomed Pharmacother ; 133: 111057, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33378962

RESUMO

Biological and prognostic roles of programmed death ligand 1 (PD-L1) remain unclear in oral squamous cell carcinoma (OSCC). Moreover, the pivotal role of tumor microenvironmental interferon-gamma (IFN-γ) in host responses to malignant cells, oral cancer growth, and PD-L1 expression has not been adequately studied. Thus, PD-L1 expression in 130 OSCC samples was analyzed using immunohistochemistry, which was found significantly overexpressed at the tumor site (P < .01). We further analyzed the effects of IFN-γ on OSCC cell proliferation using enzyme-linked immunosorbent assays and found that IFN-γ drives PD-L1 expression in OSCC cells in a dose-dependent manner. Triptolide (TPL), a bioactive compound isolated from Tripterygium wilfordii, exhibits anti-inflammatory and antitumor activities. To investigate whether the antitumor effect of TPL involves the suppression of PD-L1 expression, we treated OSCC cells in vitro and a patient-derived tumor xenograft (PDTX) model with TPL. TPL suppressed PD-L1 expression in the PDTX model, inhibiting tumor growth, and in OSCC cells in an IFN-γ-modulated microenvironment. We concluded that TPL inhibits tumor growth in oral cancer and downregulates PD-L1 expression in oral cancer cells in vitro. Our results provide evidence for the clinical development of PD-L1-targeted therapy for OSCC.


Assuntos
Antígeno B7-H1/metabolismo , Proliferação de Células/efeitos dos fármacos , Diterpenos/farmacologia , Interferon gama/farmacologia , Neoplasias Bucais/tratamento farmacológico , Fenantrenos/farmacologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Microambiente Tumoral , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antígeno B7-H1/genética , Linhagem Celular Tumoral , Regulação para Baixo , Compostos de Epóxi/farmacologia , Feminino , Humanos , Janus Quinase 2/metabolismo , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Neoplasias Bucais/imunologia , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , Carcinoma de Células Escamosas de Cabeça e Pescoço/imunologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Carga Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Nat Commun ; 11(1): 6348, 2020 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-33311506

RESUMO

Long non-coding RNAs are important regulators of biological processes including immune responses. The immunoregulatory functions of lncRNAs have been revealed primarily in murine models with limited understanding of lncRNAs in human immune responses. Here, we identify lncRNA LUCAT1 which is upregulated in human myeloid cells stimulated with lipopolysaccharide and other innate immune stimuli. Targeted deletion of LUCAT1 in myeloid cells increases expression of type I interferon stimulated genes in response to LPS. By contrast, increased LUCAT1 expression results in a reduction of the inducible ISG response. In activated cells, LUCAT1 is enriched in the nucleus where it associates with chromatin. Further, LUCAT1 limits transcription of interferon stimulated genes by interacting with STAT1 in the nucleus. Together, our study highlights the role of the lncRNA LUCAT1 as a post-induction feedback regulator which functions to restrain the immune response in human cells.


Assuntos
Regulação da Expressão Gênica , Interferons/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Animais , Fenômenos Biológicos , Cromatina/metabolismo , Citocinas/metabolismo , Retroalimentação , Técnicas de Silenciamento de Genes , Humanos , Imunidade Inata/efeitos dos fármacos , Lipopolissacarídeos/efeitos adversos , Camundongos , Células Mieloides/metabolismo , Proteínas , Fator de Transcrição STAT1/metabolismo , Células THP-1
8.
PLoS Comput Biol ; 16(12): e1008520, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33370784

RESUMO

Mycobacterium tuberculosis (Mtb) infection causes tuberculosis (TB), a disease characterized by development of granulomas. Granulomas consist of activated immune cells that cluster together to limit bacterial growth and restrict dissemination. Control of the TB epidemic has been limited by lengthy drug regimens, antibiotic resistance, and lack of a robustly efficacious vaccine. Fibrosis commonly occurs during treatment and is associated with both positive and negative disease outcomes in TB but little is known about the processes that initiate fibrosis in granulomas. Human and nonhuman primate granulomas undergoing fibrosis can have spindle-shaped macrophages with fibroblast-like morphologies suggesting a relationship between macrophages, fibroblasts, and granuloma fibrosis. This relationship has been difficult to investigate because of the limited availability of human pathology samples, the time scale involved in human TB, and overlap between fibroblast and myeloid cell markers in tissues. To better understand the origins of fibrosis in TB, we used a computational model of TB granuloma biology to identify factors that drive fibrosis over the course of local disease progression. We validated the model with granulomas from nonhuman primates to delineate myeloid cells and lung-resident fibroblasts. Our results suggest that peripheral granuloma fibrosis, which is commonly observed, can arise through macrophage-to-myofibroblast transformation (MMT). Further, we hypothesize that MMT is induced in M1 macrophages through a sequential combination of inflammatory and anti-inflammatory signaling in granuloma macrophages. We predict that MMT may be a mechanism underlying granuloma-associated fibrosis and warrants further investigation into myeloid cells as drivers of fibrotic disease.


Assuntos
Granuloma/patologia , Macrófagos/patologia , Miofibroblastos/patologia , Biologia de Sistemas , Tuberculose/patologia , Fibrose , Humanos , Mycobacterium tuberculosis/imunologia , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/metabolismo
9.
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
10.
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
11.
Life Sci ; 261: 118360, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32861799

RESUMO

AIM: Diabetic patients are reported to have a higher incidence of cataract surgery-induced retinal complications, possibly due to retinal inflammation. Our goal is to identify the key inflammatory cytokines, cells and regulatory pathways involved. MAIN METHODS: Diabetes mellitus (DM) induced by streptozotocin and control mice received extracapsular lens extraction (ECLE) in one eye. Neuroretinas were collected at postoperative day1(P1), day2(P2), and day7(P7). BV2 cells were harvested under the treatment of high glucose, lipopolysaccharide (LPS) and inhibitors. The method of qPCR, western blot and immunohistochemistry were used to identify the expression of cytokines and signaling pathways. KEY FINDINGS: ECLE induced increased inflammation in the neuroretina of surgery eye with a peak at P1. MCP-1 surge in long-term diabetes mellitus (LDM) mice at P1 is higher than short-term diabetes mellitus (SDM) mice and normal mice. Significant activation of c-jun and c-fos were found in LDM compared to normal and SDM. Advanced activation of stat1 and ERK was found at P1 in LDM instead of at P2 in SDM and Normal. Activation of microglia/macrophage was also detected in the LDM mice. Besides the inhibition of c-jun/JNK, MCP-1 expression can be attenuated by inhibiting stat1 and ERK under high glucose condition after LPS stimulation. SIGNIFICANCE: Enhancement of lens extraction-induced MCP-1 upregulation and microglia response in long-term diabetes might be due to the activation of cjun, stat1 and ERK, which provided potential therapeutic targets to attenuate retinal inflammation after surgery in diabetic individuals.


Assuntos
Extração de Catarata , Quimiocina CCL2/metabolismo , Diabetes Mellitus Experimental/genética , Sistema de Sinalização das MAP Quinases , Microglia/patologia , Proteínas Proto-Oncogênicas c-jun/metabolismo , Fator de Transcrição STAT1/metabolismo , Regulação para Cima/genética , Animais , Glucose/toxicidade , Inflamação/genética , Inflamação/patologia , Lipopolissacarídeos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/metabolismo , Retina/patologia , Regulação para Cima/efeitos dos fármacos
12.
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
13.
Viruses ; 12(8)2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32731335

RESUMO

Non-structural protein 1 (nsp1) is only characterized in alphacoronaviruses (α-CoVs) and betacoronaviruses (ß-CoVs). There have been extensive researches on how the ß-CoVs nsp1 regulates viral virulence by inhibiting host protein synthesis, but the regulatory mechanism of the α-CoVs nsp1 is still unclear. Here, we report the 2.1-Å full-length crystal structure of nsp1 in emerging porcine SADS-CoV and the 1.8-Å full-length crystal structure of nsp1 in the highly lethal cat FIPV. Although they belong to different subtypes of α-CoVs, these viruses all have a bucket-shaped fold composed of six ß-sheets, similar to the crystal structure of PEDV and TGEV nsp1. Comparing the above four structures, we found that the structure of α-CoVs nsp1 in the same subtype was more conserved. We then selected mammalian cells that were treated with SADS-CoV and FIPV nsp1 for RNA sequencing analysis and found that nsp1 had a specific inhibitory effect on interferon (IFN) and cell cycle genes. Using the Renilla luciferase (Rluc) assay and Western blotting, we confirmed that seven representative α-CoVs nsp1s could significantly inhibit the phosphorylation of STAT1-S727 and interfere with the effect of IFN-I. Moreover, the cell cycle experiment confirmed that α-CoVs nsp1 could encourage host cells to stay in the G0/G1 phase. Based on these findings, we not only greatly improved the crystal structure data on α-CoVs nsp1, but we also speculated that α-CoVs nsp1 regulated host proliferation and immune evasion-related biological functions by inhibiting the synthesis of host proteins, thus creating an environment conducive to the virus.


Assuntos
Alphacoronavirus/imunologia , Alphacoronavirus/fisiologia , Evasão da Resposta Imune/imunologia , Interferon Tipo I/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo , Alphacoronavirus/genética , Sequência de Aminoácidos , Animais , Gatos , Linhagem Celular , Cristalografia por Raios X , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Fosforilação , Estrutura Terciária de Proteína , Fator de Transcrição STAT1/metabolismo , Homologia de Sequência , Suínos , Proteínas não Estruturais Virais/genética , Replicação Viral/genética
14.
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
15.
PLoS One ; 15(7): e0228302, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32628668

RESUMO

Programmed death ligand 1 (PD-L1) has been recently shown to be a major obstacle to antiviral immunity by binding to its receptor programmed death 1 (PD-1) on specific IFN-γ producing T cells in chronic hepatitis B. Currently, IFN-α is widely used to treat hepatitis B virus (HBV) infection, but its antiviral effect vary greatly and the mechanism is not totally clear. We found that IFN-α/γ induced a marked increase of PD-L1 expression in hepatocytes. Signal and activators of transcription (Stat1) was then identified as a major transcription factor involved in IFN-α/γ-mediated PD-L1 elevation both in vitro and in mice. Blockage of the PD-L1/PD-1 interaction by a specific mAb greatly enhanced HBV-specific T cell activity by the gp96 adjuvanted therapeutic vaccine, and promoted HBV clearance in HBV transgenic mice. Our results demonstrate the IFN-α/γ-Stat1-PD-L1 axis plays an important role in mediating T cell hyporesponsiveness and inactivating liver-infiltrating T cells in the hepatic microenvironment. These data raise further potential interest in enhancing the anti-HBV efficacy of IFN-α and therapeutic vaccines.


Assuntos
Antígeno B7-H1/metabolismo , Vírus da Hepatite B/imunologia , Interferon-alfa/farmacologia , Interferon gama/farmacologia , Fator de Transcrição STAT1/metabolismo , Linfócitos T/imunologia , Regulação para Cima/efeitos dos fármacos , Animais , Anticorpos Monoclonais/uso terapêutico , Antígeno B7-H1/química , Antígeno B7-H1/genética , Antígeno B7-H1/imunologia , Sítios de Ligação , Linhagem Celular , Hepatite B/tratamento farmacológico , Hepatite B/veterinária , Antígenos de Superfície da Hepatite B/sangue , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Receptor de Morte Celular Programada 1/metabolismo , Regiões Promotoras Genéticas , Fator de Transcrição STAT1/química , Linfócitos T/metabolismo
16.
Am J Chin Med ; 48(5): 1091-1102, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32668967

RESUMO

Black ginseng (BG), which is ginseng that has been steamed and dried nine times, and its main protopanaxatriol-type ginsenosides Rg4, Rg6, Rh4, and Rg2 have been reported to exhibit various forms of biological activity, including antiseptic, antidiabetic, wound-healing, immune-stimulatory, and anti-oxidant activity. The aim of the this study was to examine the effects of [Formula: see text] (a rare protopanaxatriol-type ginsenoside fraction; Rg2, Rg4, Rg6, Rh1, and Rh4) on heme oxygenase-1 (HO-1) induction and on the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-)2 in lipopolysaccharide (LPS)-activated human pulmonary artery endothelial cells (HPAECs). [Formula: see text] was tested to determine its effect on iNOS protein expression and inflammatory markers (interleukin [IL]-1[Formula: see text] and tumor necrosis factor [TNF]-[Formula: see text] in the lung tissue of LPS-treated mice. The results showed that [Formula: see text] induced the expression of HO-1, reduced LPS-activated NF-[Formula: see text]B-luciferase activity, and inhibited iNOS/NO and COX-2/PGE2, which contributed to the inhibition of STAT-1 phosphorylation. In particular, [Formula: see text] induced the translocation of Nrf2 from the cytosol to the nucleus by increasing Nrf2-ARE activity and decreased IL-1[Formula: see text] production in LPS-activated HPAECs. This reduction in iNOS/NO expression due to [Formula: see text] was reversed by siHO-1 RNA transfection. In LPS-treated mice, [Formula: see text] significantly reduced lung tissue iNOS protein levels and TNF-[Formula: see text] levels in the bronchoalveolar lavage fluid. In conclusion, these findings indicate that [Formula: see text] has a critical anti-inflammatory effect due to its ability to regulate iNOS via the inhibition of p-STAT-1 and NF-[Formula: see text]B, and thus it may be suitable for the treatment of inflammatory disease.


Assuntos
Expressão Gênica/efeitos dos fármacos , Ginsenosídeos/farmacologia , Inflamação/genética , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Panax/química , Fator de Transcrição STAT1/metabolismo , Animais , Anti-Inflamatórios , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Ginsenosídeos/isolamento & purificação , Heme Oxigenase-1/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Inflamação/tratamento farmacológico , Mediadores da Inflamação/metabolismo , Interleucina-1beta/metabolismo , Luciferases/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Fosforilação/efeitos dos fármacos , Fitoterapia
17.
Mol Cell ; 78(6): 1207-1223.e8, 2020 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-32504554

RESUMO

Tumor interferon (IFN) signaling promotes PD-L1 expression to suppress T cell-mediated immunosurveillance. We identify the IFN-stimulated non-coding RNA 1 (INCR1) as a long noncoding RNA (lncRNA) transcribed from the PD-L1 locus and show that INCR1 controls IFNγ signaling in multiple tumor types. Silencing INCR1 decreases the expression of PD-L1, JAK2, and several other IFNγ-stimulated genes. INCR1 knockdown sensitizes tumor cells to cytotoxic T cell-mediated killing, improving CAR T cell therapy. We discover that PD-L1 and JAK2 transcripts are negatively regulated by binding to HNRNPH1, a nuclear ribonucleoprotein. The primary transcript of INCR1 binds HNRNPH1 to block its inhibitory effects on the neighboring genes PD-L1 and JAK2, enabling their expression. These findings introduce a mechanism of tumor IFNγ signaling regulation mediated by the lncRNA INCR1 and suggest a therapeutic target for cancer immunotherapy.


Assuntos
Antígeno B7-H1/genética , Interferon gama/metabolismo , RNA Longo não Codificante/genética , Idoso , Animais , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imunoterapia , Imunoterapia Adotiva/métodos , Interferon gama/genética , Interferons/genética , Interferons/metabolismo , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Pessoa de Meia-Idade , Proteína 2 Ligante de Morte Celular Programada 1/genética , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Linfócitos T Citotóxicos
18.
Viruses ; 12(6)2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-32549200

RESUMO

As more cases of COVID-19 are studied and treated worldwide, it had become apparent that the lethal and most severe cases of pneumonia are due to an out-of-control inflammatory response to the SARS-CoV-2 virus. I explored the putative causes of this specific feature through a detailed genomic comparison with the closest SARS-CoV-2 relatives isolated from bats, as well as previous coronavirus strains responsible for the previous epidemics (SARS-CoV and MERS-CoV). The high variability region of the nsp3 protein was confirmed to exhibit the most variations between closest strains. It was then studied in the context of physiological and molecular data available in the literature. A number of convergent findings suggest de-mono-ADP-ribosylation (de-MARylation) of STAT1 by the SARS-CoV-2 nsp3 as a putative cause of the cytokine storm observed in the most severe cases of COVID-19. This may suggest new therapeutic approaches and help in designing assays to predict the virulence of naturally circulating SARS-like animal coronaviruses.


Assuntos
ADP-Ribosilação/fisiologia , Betacoronavirus/genética , Síndrome da Liberação de Citocina/patologia , Fator de Transcrição STAT1/metabolismo , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos/genética , Infecções por Coronavirus/patologia , Humanos , Inflamação/patologia , Inflamação/virologia , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Pandemias , Peptidil Dipeptidase A/biossíntese , Peptidil Dipeptidase A/genética , Pneumonia Viral/patologia , Vírus da SARS/genética , Homologia de Sequência , Proteínas não Estruturais Virais/genética
19.
Biochem Pharmacol ; 180: 114122, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32592721

RESUMO

An unprecedented biological function of natural cardenolides independent of their membrane target Na+/K+-ATPase is disclosed. Previously, we reported that cardenolides impart anti-transmissible gastroenteritis coronavirus (anti-TGEV) activity through the targeting of Na+/K+-ATPase and its associated PI3K_PDK1_RSK2 signaling. Swine testis cells with Na+/K+-ATPase α1 knocked down exhibited decreased susceptibility to TGEV infectivity and attenuated PI3K_PDK1_RSK2 signaling. Herein, we further explored a Na+/K+-ATPase-independent signaling axis induced by natural cardenolides that also afforded significant anti-coronaviral activity for porcine TGEV and human HCoV-OC43. Using pharmacological inhibition and gene silencing techniques, we found that this anti-TGEV or anti-HCoV-OC43 activity was caused by JAK1 proteolysis and mediated through upstream activation of Ndfip1/2 and its effector NEDD4. This study provides novel insights into the pharmacological effects of natural cardenolides, and is expected to inform their future development as antiviral agents.


Assuntos
Antivirais/farmacologia , Cardenolídeos/farmacologia , Coronavirus Humano OC43/efeitos dos fármacos , Janus Quinase 1/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Vírus da Gastroenterite Transmissível/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular , Regulação para Baixo/efeitos dos fármacos , Feminino , Técnicas de Silenciamento de Genes , Humanos , Leupeptinas , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Ouabaína/farmacologia , Fosforilação , Inibidores de Proteases/farmacologia , Proteólise , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Suínos
20.
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
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