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1.
Front Immunol ; 12: 745802, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34671360

RESUMO

Immune modulation for the treatment of chronic hepatitis B (CHB) has gained more traction in recent years, with an increasing number of compounds designed for targeting different host pattern recognition receptors (PRRs). These agonistic molecules activate the receptor signaling pathway and trigger an innate immune response that will eventually shape the adaptive immunity for control of chronic infection with hepatitis B virus (HBV). While definitive recognition of HBV nucleic acids by PRRs during viral infection still needs to be elucidated, several viral RNA sensing receptors, including toll-like receptors 7/8/9 and retinoic acid inducible gene-I-like receptors, are explored preclinically and clinically as possible anti-HBV targets. The antiviral potential of viral DNA sensing receptors is less investigated. In the present study, treatment of primary woodchuck hepatocytes generated from animals with CHB with HSV-60 or poly(dA:dT) agonists resulted in increased expression of interferon-gamma inducible protein 16 (IFI16) or Z-DNA-binding protein 1 (ZBP1/DAI) and absent in melanoma 2 (AIM2) receptors and their respective adaptor molecules and effector cytokines. Cytosolic DNA sensing receptor pathway activation correlated with a decline in woodchuck hepatitis virus (WHV) replication and secretion in these cells. Combination treatment with HSV-60 and poly(dA:dT) achieved a superior antiviral effect over monotreatment with either agonist that was associated with an increased expression of effector cytokines. The antiviral effect, however, could not be enhanced further by providing additional type-I interferons (IFNs) exogenously, indicating a saturated level of effector cytokines produced by these receptors following agonism. In WHV-uninfected woodchucks, a single poly(dA:dT) dose administered via liver-targeted delivery was well-tolerated and induced the intrahepatic expression of ZBP1/DAI and AIM2 receptors and their effector cytokines, IFN-ß and interleukins 1ß and 18. Receptor agonism also resulted in increased IFN-γ secretion of peripheral blood cells. Altogether, the effect on WHV replication and secretion following in vitro activation of IFI16, ZBP1/DAI, and AIM2 receptor pathways suggested an antiviral benefit of targeting more than one cytosolic DNA receptor. In addition, the in vivo activation of ZBP1/DAI and AIM2 receptor pathways in liver indicated the feasibility of the agonist delivery approach for future evaluation of therapeutic efficacy against HBV in woodchucks with CHB.


Assuntos
Antivirais/farmacologia , Vírus da Hepatite B da Marmota/efeitos dos fármacos , Hepatite B/tratamento farmacológico , Hepatócitos/efeitos dos fármacos , Poli dA-dT/farmacologia , Receptores de Superfície Celular/agonistas , Receptores de Reconhecimento de Padrão/agonistas , Receptores Virais/agonistas , Animais , Antivirais/uso terapêutico , Células Cultivadas , Citocinas/biossíntese , Citocinas/genética , Citosol/virologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Sinergismo Farmacológico , Hepatite B/imunologia , Hepatite B/virologia , Vírus da Hepatite B da Marmota/fisiologia , Hepatócitos/virologia , Imunidade Inata , Interferons/farmacologia , Fígado/efeitos dos fármacos , Fígado/virologia , Marmota , Infecção Persistente , Poli dA-dT/uso terapêutico , Pteridinas/farmacologia , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/genética , Receptores de Reconhecimento de Padrão/biossíntese , Receptores de Reconhecimento de Padrão/genética , Receptores Virais/biossíntese , Receptores Virais/genética , Replicação Viral/efeitos dos fármacos
2.
Mol Med Rep ; 23(5)2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33760111

RESUMO

Cholesteatoma constitutes an acquired benign epidermal non­permanent bone lesion that is locally destructive and patients often relapse. Inflammasomes, which mediate the maturation and production of IL­18 and IL­1ß, resulting in pyroptosis, have been documented to serve a core function in multiple inflammatory conditions. Absent in melanoma 2 (AIM2) is an inflammasome that identifies cytoplasmic DNA and has previously been reported as a pivotal modulator of inflammatory responses. Therefore, the present study aimed to determine the expression levels of AIM2 in human cholesteatoma tissues, and elucidate its function in modulating cytokine production. The expression levels of IL­18, apoptosis­associated speck­like protein containing a CARD (ASC), IL­1ß, AIM2 and caspase­1 were markedly elevated in cholesteatoma tissues. Protein expression levels of AIM2, caspase­1 and ASC were localized in the cellular cytoplasm, primarily in the granular and prickle­cell layers in the cholesteatoma epithelium. Induction using IFN­Î³, as well as cytoplasmic DNA markedly activated the AIM2 inflammasome and elevated the release of IL­18 and IL­1ß in human cholesteatoma keratinocytes. IFN­Î³ was found to enhance poly(dA:dT)­induced pyroptosis of cells and cytokine production. The results of the present study revealed that AIM2 expressed in human cholesteatoma serves a vital function in the inflammatory response by initiating the inflammasome signaling cascade in cholesteatoma.


Assuntos
Neoplasias Ósseas/genética , Colesteatoma/genética , Proteínas de Ligação a DNA/genética , Interleucina-18/genética , Interleucina-1beta/genética , Animais , Apoptose/efeitos dos fármacos , Neoplasias Ósseas/patologia , Proteínas Adaptadoras de Sinalização CARD/genética , Caspase 1/genética , Colesteatoma/patologia , Citocinas/biossíntese , Citocinas/genética , Citoplasma/genética , DNA/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamassomos/genética , Interferon gama/genética , Interleucina-18/biossíntese , Interleucina-1beta/biossíntese , Queratinócitos/metabolismo , Neoplasias/genética , Neoplasias/patologia , Poli dA-dT/farmacologia , Piroptose/efeitos dos fármacos , Piroptose/genética
3.
Front Immunol ; 11: 598884, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33664729

RESUMO

Epithelial cells of the female reproductive tract (FRT) participate in the initial innate immunity against viral infections. Poly(dA:dT) is a synthetic analog of B form double-stranded (ds) DNA which can activate the interferon (IFN) signaling pathway-mediated antiviral immunity through DNA-dependent RNA Polymerase III. Here we investigated whether poly(dA:dT) could inhibit herpes simplex virus type 2 (HSV-2) infection of human cervical epithelial cells (End1/E6E7). We demonstrated that poly(dA:dT) treatment of End1/E6E7 cells could significantly inhibit HSV-2 infection. Mechanistically, poly(dA:dT) treatment of the cells induced the expression of the intracellular IFNs and the multiple antiviral IFN-stimulated genes (ISGs), including IFN-stimulated gene 15 (ISG15), IFN-stimulated gene 56 (ISG56), 2'-5'-oligoadenylate synthetase 1 (OAS1), 2'-5'-oligoadenylate synthetase 2 (OAS2), myxovirus resistance protein A (MxA), myxovirus resistance protein B (MxB), virus inhibitory protein, endoplasmic reticulum-associated, IFN-inducible (Viperin), and guanylate binding protein 5 (GBP5). Further investigation showed that the activation of RIG-I was largely responsible for poly(dA:dT)-mediated HSV-2 inhibition and IFN/ISGs induction in the cervical epithelial cells, as RIG-I knockout abolished the poly(dA:dT) actions. These observations demonstrate the importance for design and development of AT-rich dsDNA-based intervention strategies to control HSV-2 mucosal transmission in FRT.


Assuntos
Colo do Útero/metabolismo , Colo do Útero/virologia , Proteína DEAD-box 58/metabolismo , Herpes Genital/metabolismo , Herpes Genital/virologia , Herpesvirus Humano 2/efeitos dos fármacos , Herpesvirus Humano 2/fisiologia , Poli dA-dT/farmacologia , Receptores Imunológicos/metabolismo , Biomarcadores , Linhagem Celular , Sobrevivência Celular , Proteína DEAD-box 58/genética , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Feminino , Técnicas de Silenciamento de Genes , Herpes Genital/tratamento farmacológico , Humanos , Imunofenotipagem , Janus Quinases/metabolismo , Mucosa/metabolismo , Mucosa/virologia , Receptores Imunológicos/genética , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
4.
Biochem Biophys Res Commun ; 522(4): 939-944, 2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-31806367

RESUMO

The retinoic-acid inducible gene (RIG)-I is a cytoplasmic pattern recognition receptor that senses single-stranded (ss) or double-stranded (ds) RNA. RIG-I also senses AT-rich dsDNA, poly(dA:dT), through the action of an RNA polymerase III-transcribed RNA intermediate. Upon the binding of an RNA ligand, RIG-I binds to the mitochondrial antiviral-signaling protein (MAVS) and induces the formation of filamentous aggregates of MAVS, leading to the formation of a signaling complex that drives Type I interferon (IFN) responses. In the current study, we investigated the issue of whether the SUMOylation of MAVS induced by poly(dA:dT) affects the aggregation of MAVS in the RIG-I/MAVS pathway in human keratinocytes. Our results show that the poly(dA:dT)-induced secretion of IFN-ß was dependent on RIG-I and MAVS. The inhibition of SUMOylation by Ginkgolic acid or Ubc9 siRNA was found to inhibit the poly(dA:dT)-induced secretion of IFN-ß, suggesting that the SUMOylation is required for the poly(dA:dT)-activated RIG-I/MAVS pathway, which drives the secretion of IFN-ß. In addition, treatment with poly(dA:dT) enhanced the formation of polymeric chains of small-ubiquitin like modifiers (SUMO)3, but not SUMO1 and SUMO2, on MAVS. Our results also show that the conjugation of SUMO3 to MAVS induced by poly (dA:dT) enhanced the aggregation of MAVS. These collective results show that the formation of SUMO3-conjugated chains of MAVS induced by poly (dA:dT), a ligand of RIG-I, enhances the aggregation of MAVS which, in turn, drives the secretion of IFN-ß in human keratinocytes.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína DEAD-box 58/metabolismo , Interferon beta/metabolismo , Queratinócitos/metabolismo , Poli dA-dT/farmacologia , Agregados Proteicos , Ubiquitinas/metabolismo , Linhagem Celular , Humanos , Queratinócitos/efeitos dos fármacos , Ligantes , Agregados Proteicos/efeitos dos fármacos , Domínios Proteicos , RNA Interferente Pequeno/metabolismo , Receptores Imunológicos , Salicilatos/farmacologia , Deleção de Sequência , Sumoilação/efeitos dos fármacos , Enzimas de Conjugação de Ubiquitina/metabolismo
5.
Biochem Biophys Res Commun ; 503(1): 116-122, 2018 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-29857000

RESUMO

Quercetin, a polyphenol, belongs to a class of flavonoids that exerts anti-inflammatory effects. Interleukin (IL)-18 is a member of the IL-1 family cytokine that regulates immune responses and is implicated in various inflammatory skin diseases. Absent in melanoma 2 (AIM2) is a cytosolic double-stranded (ds) DNA sensor that recognizes the dsDNA of a microbial or host origin. Binding of dsDNA to AIM2 simulates caspase-1-dependent inflammasome activity, which leads to the production of IL-1ß and IL-18. Increased levels of AIM2 have been observed in patients with inflammatory skin diseases. In the current study, we investigated the issue of whether or how Quercetin attenuates poly (dA:dT), a synthetic analog of microbial dsDNA, -induced IL-18 secretion in IFN-γ-primed human keratinocytes. Treatment with 5 and 10 µM of Quercetin inhibited the poly (dA:dT)-induced secretion of IL-18 after IFN-γ priming and before poly (dA:dT)-induced AIM2 activation. In addition, treatment with Quercetin at 10 µM, significantly inhibited the phosphorylation of JAK2 and STAT1, and the nuclear translocation of phosphorylated STAT1 in poly (dA:dT)-treated and IFN-γ-primed keratinocytes. These results suggest that treatment with Quercetin inhibits the poly (dA:dT)-induced secretion of IL-18 via down-regulation of the expressions of AIM2 and pro-caspase-1 by inhibiting the JAK2/STAT1 pathway in IFN-γ-primed keratinocytes.


Assuntos
Caspase 1/metabolismo , Proteínas de Ligação a DNA/metabolismo , Interleucina-18/biossíntese , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Quercetina/farmacologia , Anti-Inflamatórios não Esteroides/farmacologia , Caspase 1/genética , Linhagem Celular , Células Cultivadas , Proteínas de Ligação a DNA/genética , Regulação para Baixo/efeitos dos fármacos , Humanos , Interferon gama/metabolismo , Interferon gama/farmacologia , Janus Quinase 2/metabolismo , Queratinócitos/imunologia , Poli dA-dT/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais/efeitos dos fármacos
6.
Int J Mol Sci ; 19(3)2018 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-29518010

RESUMO

Keratinocytes are non-professional immune cells contributing actively to innate immune responses partially by reacting to a wide range of molecular patterns by activating pattern recognition receptors. Cytosolic nucleotide fragments as pathogen- or self-derived trigger factors are activating inflammasomes and inducing anti-viral signal transduction pathways as well as inducing expression of inflammatory cytokines. We aimed to compare the induced inflammatory reactions in three keratinocyte cell types-normal human epidermal keratinocytes, the HaCaT cell line and the HPV-KER cell line-upon exposure to the synthetic RNA and DNA analogues poly(I:C) and poly(dA:dT) to reveal the underlying signaling events. Both agents induced the expression of interleukin-6 and tumor necrosis factor α in all cell types; however, notable kinetic and expression level differences were found. Western blot analysis revealed rapid activation of the nuclear factor κB (NF-κB), mitogen activated protein kinase and signal transducers of activator of transcription (STAT) signal transduction pathways in keratinocytes upon poly(I:C) treatment, while poly(dA:dT) induced slower activation. Inhibition of NF-κB, p38, STAT-1 and STAT-3 signaling resulted in decreased cytokine expression, whereas inhibition of mitogen-activated protein kinase kinase 1/2 (MEK1/2) signaling showed a negative feedback role in both poly(I:C)- and poly(dA:dT)-induced cytokine expression. Based on our in vitro results nucleotide fragments are able to induce inflammatory reactions in keratinocytes, but with different rate and kinetics of cytokine expression, explained by faster activation of signaling routes by poly(I:C) than poly(dA:dT).


Assuntos
Queratinócitos/metabolismo , Poli dA-dT/farmacologia , Linhagem Celular , Células Cultivadas , Citocinas/metabolismo , Humanos , Inflamassomos/metabolismo , Queratinócitos/efeitos dos fármacos , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/metabolismo , NF-kappa B/metabolismo , Fatores de Transcrição STAT/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
7.
Proc Natl Acad Sci U S A ; 114(7): E1062-E1071, 2017 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-28137853

RESUMO

The ring-shaped cohesin complex orchestrates long-range DNA interactions to mediate sister chromatid cohesion and other aspects of chromosome structure and function. In the yeast Saccharomyces cerevisiae, the complex binds discrete sites along chromosomes, including positions within and around genes. Transcriptional activity redistributes the complex to the 3' ends of convergently oriented gene pairs. Despite the wealth of information about where cohesin binds, little is known about cohesion at individual chromosomal binding sites and how transcription affects cohesion when cohesin complexes redistribute. In this study, we generated extrachromosomal DNA circles to study cohesion in response to transcriptional induction of a model gene, URA3. Functional cohesin complexes loaded onto the locus via a poly(dA:dT) tract in the gene promoter and mediated cohesion before induction. Upon transcription, the fate of these complexes depended on whether the DNA was circular or not. When gene activation occurred before DNA circularization, cohesion was lost. When activation occurred after DNA circularization, cohesion persisted. The presence of a convergently oriented gene also prevented transcription-driven loss of functional cohesin complexes, at least in M phase-arrested cells. The results are consistent with cohesin binding chromatin in a topological embrace and with transcription mobilizing functional complexes by sliding them along DNA.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos Fúngicos/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas de Saccharomyces cerevisiae/metabolismo , Ativação Transcricional/fisiologia , Adenosina Trifosfatases/metabolismo , Sítios de Ligação , Cromossomos Fúngicos/ultraestrutura , DNA Circular/metabolismo , DNA Fúngico/genética , Proteínas de Ligação a DNA/metabolismo , Herança Extracromossômica , Genes Fúngicos , Genes Reporter , Genes Sintéticos , Metáfase , Complexos Multiproteicos/metabolismo , Poli dA-dT/farmacologia , Regiões Promotoras Genéticas/genética , Ligação Proteica , Sequências Reguladoras de Ácido Nucleico , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Coesinas
8.
Virus Res ; 232: 13-21, 2017 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-28119118

RESUMO

The cellular antiviral innate immune system is essential for host defense and viruses have evolved a variety of strategies to evade the innate immunity. Human T lymphotropic virus type 1 (HTLV-1) belongs to the deltaretrovirus family and it can establish persistent infection in human beings for many years. However, how this virus evades the host innate immune responses remains unclear. Here we report a new strategy used by HTLV-1 to block innate immune responses. We observed that stimulator of interferon genes (STING) limited HTLV-1 protein expression and was critical to HTLV-1 reverse transcription intermediate (RTI) ssDNA90 triggered interferon (IFN)-ß production in phorbol12-myristate13-acetate (PMA)-differentiated THP1 (PMA-THP1) cells. The HTLV-1 protein Tax inhibited STING overexpression induced transcriptional activation of IFN-ß. Tax also impaired poly(dA:dT), interferon stimulatory DNA (ISD) or cyclic GMP-AMP (cGAMP) -stimulated IFN-ß production, which was dependent on STING activation. Coimmunoprecipitation assays and confocal microscopy indicated that Tax was associated with STING in the same complex. Mechanistic studies suggested that Tax decreased the K63-linked ubiquitination of STING and disrupted the interactions between STING and TANK-binding kinase 1 (TBK1). These findings may shed more light on the molecular mechanisms underlying HTLV-1 infection.


Assuntos
Produtos do Gene tax/genética , Vírus Linfotrópico T Tipo 1 Humano/genética , Evasão da Resposta Imune , Proteínas de Membrana/genética , Proteínas Serina-Treonina Quinases/genética , Regulação da Expressão Gênica , Produtos do Gene tax/imunologia , Vírus Linfotrópico T Tipo 1 Humano/imunologia , Humanos , Imunidade Inata , Interferon beta/genética , Interferon beta/imunologia , Lisina/metabolismo , Ativação de Macrófagos/efeitos dos fármacos , Proteínas de Membrana/imunologia , Nucleotídeos Cíclicos/farmacologia , Poli dA-dT/farmacologia , Ligação Proteica , Proteínas Serina-Treonina Quinases/imunologia , Transcrição Reversa , Transdução de Sinais , Células THP-1 , Acetato de Tetradecanoilforbol/farmacologia , Ubiquitinação
9.
Cytotherapy ; 17(10): 1332-41, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26227206

RESUMO

BACKGROUND AIMS: Previously, we showed that human mesenchymal stromal cells (hMSCs) were activated to express tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) upon TNF-α stimulation, induced cell death in triple-negative breast cancer (TNBC) MDA-MB-231 cells (MDA), and RNA released from apoptotic MDA further increased TRAIL expression in hMSCs. This feed-forward stimulation increased apoptosis in MDA cells. Here, we tested whether TRAIL-expressing hMSCs, in combination with a sub-toxic-dose of a chemotherapy drug doxorubicin, would overcome TRAIL resistance and create synergistic effects on targeting metastatic TNBC. METHODS: To optimize conditions for the combination treatment, we (i) selected an optimal condition to activate hMSCs for TRAIL expression, (ii) selected an optimal dose of doxorubicin treatment, (iii) examined underlying mechanisms in vitro and (iv) tested the efficacy of the optimized conditions in a xenograft mouse model of human breast cancer lung metastasis. RESULTS: The results showed that DNA fragments from apoptotic MDA triggered hMSCs to increase further TRAIL expression in an absent in melanoma 2 (AIM2)-dependent manner, and thus higher TRAIL-expressing hMSCs stimulated with synthetic DNA, poly(deoxyadenylic-deoxythymidylic) acid [poly(dA:dT)], more effectively suppressed tumor progression in vivo. Furthermore, activated hMSCs increased apoptosis in MDA cells when combined with a sub-toxic dose of doxorubicin, which was mediated by up-regulating TRAIL and Fas-related pathways. When we combined the optimized conditions, pre-activated hMSCs with poly (dA:dT) synergistically reduced tumor burden even with minimal doxorubicin treatment in a xenograft mouse model of human breast cancer lung metastasis. CONCLUSIONS: These results suggest that the treatment of hMSCs with a sub-toxic dose of doxorubicin can overcome TRAIL resistance and be a potential novel therapy for TNBC metastasis treatment.


Assuntos
Apoptose , Doxorrubicina/uso terapêutico , Transplante de Células-Tronco Mesenquimais , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Neoplasias de Mama Triplo Negativas/terapia , Animais , Linhagem Celular Tumoral , RNA Helicases DEAD-box/genética , Fragmentação do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Humanos , Helicase IFIH1 Induzida por Interferon , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Células-Tronco Mesenquimais/metabolismo , Camundongos , Poli dA-dT/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/biossíntese , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologia , Fator de Necrose Tumoral alfa/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
10.
J Hypertens ; 33(8): 1658-65, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26002845

RESUMO

OBJECTIVES: Preeclampsia is a serious pregnancy-specific hypertensive syndrome that is characterized by widespread maternal endothelial dysfunction. Previous studies have shown that increased levels of circulating cell-free fetal DNA in women with preeclampsia correspond to the degree of disease severity; however, it is unknown whether this DNA is a key signal that contributes to the development of preeclampsia. The detection of DNA is critical to appropriate innate immune responses. The interferon-inducible protein 16 (IFI16) - a member of the HIN-200 family - is an innate immune receptor for intracellular DNA, which is implicated in the control of cell growth, apoptosis, angiogenesis, and immunomodulation; however, its role in preeclampsia remains unresolved. Here, we tested the hypothesis that this DNA can activate IFI16 in the placentas of women with preeclampsia and is sufficient to induce soluble fms-like tyrosine kinase 1 (sFlt-1) and soluble endoglin (sEng) production. METHODS: We characterized IFI16 in severe preeclamptic placentas and assessed whether DNA increased the release of sFlt-1 and sEng from trophoblast cells and placental explants. Furthermore, we determined whether IFI16 was involved in DNA-induced sFlt-1 and sEng production. RESULTS: Placental immunoreactivity and protein levels of IFI16 were significantly increased in women with preeclampsia compared to matched control women. Treatment of human trophoblasts with the IFI16 agonist poly(dA:dT) significantly increased IFI16 levels. Furthermore, poly(dA:dT) induced sFlt-1 and sEng production by human trophoblasts in an IFI16-dependent manner. CONCLUSIONS: We conclude that trophoblast cells respond to cell-free fetal DNA through the IFI16 receptor, resulting in the production of the preeclampsia-related antiangiogenic factors sFlt-1 and sEng.


Assuntos
Antígenos CD/biossíntese , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Placenta/metabolismo , Pré-Eclâmpsia/metabolismo , Receptores de Superfície Celular/biossíntese , Trofoblastos/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Adulto , Células Cultivadas , Endoglina , Feminino , Humanos , Fosforilação , Placenta/imunologia , Poli dA-dT/farmacologia , Gravidez , Biossíntese de Proteínas/efeitos dos fármacos , Transdução de Sinais , Trofoblastos/efeitos dos fármacos
11.
Mol Immunol ; 65(2): 436-45, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25765883

RESUMO

Cyclic GMP-AMP synthase (cGAS), which belongs to the nucleotidyltransferase family, recognizes cytosolic DNA and induces the type I interferon (IFN) pathway through the synthesis of the second messenger cGAMP. In this study, porcine cGAS (p-cGAS) was identified and its tissue distribution, subcellular localization, and functions in innate immunity were characterized. The coding sequence of p-cGAS is 1494 bp long, encodes 497 amino acids, and is most similar (74%) to Bos taurus cGAS. p-cGAS mRNA is abundant in the spleen, duodenum, jejunum, and ileum. The subcellular distribution of p-cGAS is not only in the cytosol, but also on the endoplasmic reticulum (ER) membrane. The overexpression of wild-type p-cGAS in porcine kidney epithelial cells, but not its catalytically inactive mutants, induced IFN-ß expression, which was dependent on STING and IRF3. However, the downregulation of p-cGAS by RNA interference markedly reduced IFN-ß expression after pseudorabies virus (PRV) infection or poly(dA:dT) transfection. These results demonstrate that p-cGAS is an important DNA sensor, required for IFN-ß activation.


Assuntos
Retículo Endoplasmático , Regulação da Expressão Gênica/imunologia , Membranas Intracelulares/imunologia , Nucleotidiltransferases , Suínos , Sequência de Aminoácidos , Animais , Bovinos , Clonagem Molecular , Retículo Endoplasmático/genética , Retículo Endoplasmático/imunologia , Interferon beta/genética , Interferon beta/imunologia , Dados de Sequência Molecular , Mutação , Nucleotidiltransferases/genética , Nucleotidiltransferases/imunologia , Especificidade de Órgãos/genética , Especificidade de Órgãos/imunologia , Poli dA-dT/farmacologia , Homologia de Sequência de Aminoácidos , Suínos/genética , Suínos/imunologia
12.
Acta Derm Venereol ; 95(2): 140-6, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24909845

RESUMO

Narrow-band UVB (NB-UVB) phototherapy is commonly used for treatment of psoriasis, though the mechanisms underlying its efficacy have not been completely elucidated. We used gene expression profiling to characterise gene expression in lesional epidermis from psoriasis patients in the middle and late stages of NB-UVB photo-therapy. Increased melanogenesis gene expression was the earliest response to phototherapy. At the end of treatment, genes responding to phototherapy and correlated to treatment outcome were involved in oxidation reduction, growth and mitochondria organisation. Particularly, SPATA18, a key regulator of mitochondrial quality, was significantly down-regulated in psoriasis (p < 0.05). Poly(dA:dT) and poly(I:C) stimulation increased SPATA18 level in primary keratinocytes, indicating the importance of mitochondria quality control under innate immune induced oxidative stress. Normalised SPATA18 expression after phototherapy indicates improved mitochondrial quality control and restored cellular redox status. Our data suggest that oxidation reduction is critical for the resolution of psoriatic plaques following NB-UVB phototherapy.


Assuntos
Epiderme/efeitos da radiação , Queratinócitos/efeitos da radiação , Psoríase/radioterapia , Terapia Ultravioleta/métodos , Biópsia , Linhagem Celular , Análise Discriminante , Epiderme/imunologia , Epiderme/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/imunologia , Queratinócitos/metabolismo , Análise dos Mínimos Quadrados , Melaninas/biossíntese , Mitocôndrias/imunologia , Mitocôndrias/metabolismo , Mitocôndrias/efeitos da radiação , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Oxirredução , Poli I-C/farmacologia , Poli dA-dT/farmacologia , Psoríase/genética , Psoríase/imunologia , Psoríase/metabolismo , Fatores de Tempo , Resultado do Tratamento
13.
PLoS One ; 9(7): e102033, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25025687

RESUMO

X-linked severe combined immunodeficiency (XSCID) is caused by a genetic mutation within the common gamma chain (γc), an essential component of the cytokine receptors for interleukin (IL)-2, IL-4, IL-7, IL-9, IL-15, and IL-21. XSCID patients are most commonly treated with bone marrow transplants (BMT) to restore systemic immune function. However, BMT-XSCID humans and dogs remain at an increased risk for development of cutaneous papillomavirus (PV) infections and their associated neoplasms, most typically cutaneous papillomas. Since basal keratinocytes are the target cell for the initial PV infection, we wanted to determine if canine XSCID keratinocytes have a diminished antiviral cytokine response to poly(dA:dT) and canine papillomavirus-2 (CPV-2) upon initial infection. We performed quantitative RT-PCR for antiviral cytokines and downstream interferon stimulated genes (ISG) on poly(dA:dT) stimulated and CPV-2 infected monolayer keratinocyte cultures derived from XSCID and normal control dogs. We found that XSCID keratinocytes responded similarly to poly(dA:dT) as normal keratinocytes by upregulating antiviral cytokines and ISGs. CPV-2 infection of both XSCID and normal keratinocytes did not result in upregulation of antiviral cytokines or ISGs at 2, 4, or 6 days post infection. These data suggest that the antiviral response to initial PV infection of basal keratinocytes is similar between XSCID and normal patients, and is not the likely source for the remaining immunodeficiency in XSCID patients.


Assuntos
Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Infecções por Papillomavirus/etiologia , Poli dA-dT/farmacologia , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/imunologia , Animais , Sequência de Bases , Transplante de Medula Óssea , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Cães , Feminino , Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Subunidade gama Comum de Receptores de Interleucina/química , Subunidade gama Comum de Receptores de Interleucina/genética , Queratinócitos/virologia , Dados de Sequência Molecular , Mutação , Papillomaviridae , Infecções por Papillomavirus/tratamento farmacológico , Poli dA-dT/administração & dosagem , Cultura Primária de Células , RNA Mensageiro/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/complicações , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/terapia
14.
J Cereb Blood Flow Metab ; 34(4): 621-9, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24398937

RESUMO

The central nervous system (CNS) is an active participant in the innate immune response to infection and injury. In these studies, we show embryonic cortical neurons express a functional, deoxyribonucleic acid (DNA)-responsive, absent in melanoma 2 (AIM2) inflammasome that activates caspase-1. Neurons undergo pyroptosis, a proinflammatory cell death mechanism characterized by the following: (a) oligomerization of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC); (b) caspase-1 dependency; (c) formation of discrete pores in the plasma membrane; and (d) release of the inflammatory cytokine interleukin-1ß (IL-1ß). Probenecid and Brilliant Blue FCF, inhibitors of the pannexin1 channel, prevent AIM2 inflammasome-mediated cell death, identifying pannexin1 as a cell death effector during pyroptosis and probenecid as a novel pyroptosis inhibitor. Furthermore, we show activation of the AIM2 inflammasome in neurons by cerebrospinal fluid (CSF) from traumatic brain injury (TBI) patients and oligomerization of ASC. These findings suggest neuronal pyroptosis is an important cell death mechanism during CNS infection and injury that may be attenuated by probenecid.


Assuntos
Apoptose , Inflamassomos/metabolismo , Neurônios/metabolismo , Proteínas Nucleares/metabolismo , Adolescente , Adulto , Idoso , Animais , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Proteínas Reguladoras de Apoptose/metabolismo , Lesões Encefálicas/líquido cefalorraquidiano , Lesões Encefálicas/imunologia , Lesões Encefálicas/metabolismo , Lesões Encefálicas/patologia , Caspase 1/metabolismo , Técnicas de Cultura de Células , Morte Celular , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Proteínas de Ligação a DNA , Feminino , Humanos , Imunidade Inata/efeitos dos fármacos , Inflamassomos/imunologia , Masculino , Pessoa de Meia-Idade , Neurônios/efeitos dos fármacos , Neurônios/imunologia , Neurônios/patologia , Poli dA-dT/farmacologia , Probenecid/farmacologia , Ratos , Ratos Sprague-Dawley , Adulto Jovem
15.
Vet Immunol Immunopathol ; 153(3-4): 177-86, 2013 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-23557936

RESUMO

Papillomaviruses (PV) are double stranded (ds) DNA viruses that infect epithelial cells within the skin or mucosa, most often causing benign neoplasms that spontaneously regress. The immune system plays a key role in the defense against PVs. Since these viruses infect keratinocytes, we wanted to investigate the role of the keratinocyte in initiating an immune response to canine papillomavirus-2 (CPV-2) in the dog. Keratinocytes express a variety of pattern recognition receptors (PRR) to distinguish different cutaneous pathogens and initiate an immune response. We examined the mRNA expression patterns for several recently described cytosolic nucleic acid sensing PRRs in canine monolayer keratinocyte cultures using quantitative reverse transcription-polymerase chain reaction. Unstimulated normal cells were found to express mRNA for melanoma differentiation associated gene 5 (MDA5), retinoic acid-inducible gene I (RIG-I), DNA-dependent activation of interferon regulatory factors, leucine rich repeat flightless interacting protein 1, and interferon inducible gene 16 (IFI16), as well as their adaptor molecules myeloid differentiation primary response gene 88, interferon-ß promoter stimulator 1, and endoplasmic reticulum-resident transmembrane protein stimulator of interferon genes. When stimulated with synthetic dsDNA [poly(dA:dT)] or dsRNA [poly(I:C)], keratinocytes responded with increased mRNA expression levels for interleukin-6, tumor necrosis factor-α, interferon-ß, RIG-I, IFI16, and MDA5. There was no detectable increase in mRNA expression, however, in keratinocytes infected with CPV-2. Furthermore, CPV-2-infected keratinocytes stimulated with poly(dA:dT) and poly(I:C) showed similar mRNA expression levels for these gene products when compared with expression levels in uninfected cells. These results suggest that although canine keratinocytes contain functional PRRs that can recognize and respond to dsDNA and dsRNA ligands, they do not appear to recognize or initiate a similar response to CPV-2.


Assuntos
Citocinas/genética , Interferon Tipo I/genética , Queratinócitos/imunologia , Papillomaviridae/imunologia , Poli dA-dT/farmacologia , Animais , Células Cultivadas , Cães , Queratinócitos/efeitos dos fármacos , RNA Mensageiro/análise , Receptores de Reconhecimento de Padrão/genética , Regulação para Cima/efeitos dos fármacos
16.
Mol Cell Proteomics ; 11(11): 1230-44, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22869553

RESUMO

Inflammasomes are cytoplasmic receptors that can recognize intracellular pathogens or danger signals and are critical for interleukin 1ß production. Although several key components of inflammasome activation have been identified, there has not been a systematic analysis of the protein components found in the stimulated complex. In this study, we used the isobaric tags for relative and absolute quantification approach to systemically analyze the interactomes of the NLRP3, AIM2, and RIG-I inflammasomes in nasopharyngeal carcinoma cells treated with specific stimuli of these interactomes (H2O2, poly (dA:dT), and EBV noncoding RNA, respectively). We identified a number of proteins that appeared to be involved in the interactomes and also could be precipitated with anti-apoptosis-associated speck-like protein containing caspase activation and recruitment domain antibodies after stimulation. Among them, end binding protein 1 was an interacting component in all three interactomes. Silencing of end binding protein 1 expression by small interfering RNA inhibited the activation of the three inflammasomes, as indicated by reduced levels of interleukin 1ß secretion. We confirmed that end binding protein 1 directly interacted with AIM2 and ASC in vitro and in vivo. Most importantly, fluorescence confocal microscopy showed that end binding protein 1 was required for formation of the speck-like particles that represent activation of the AIM2 inflammasome. In nasopharyngeal carcinoma tissues, immunohistochemical staining showed that end binding protein 1 expression was elevated and significantly correlated with AIM2 and ASC expression in nasopharyngeal carcinoma tumor cells. In sum, we profiled the interactome components of three inflammasomes and show for the first time that end binding protein 1 is crucial for the speck-like particle formation that represents activated inflammasomes.


Assuntos
Inflamassomos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Adolescente , Adulto , Idoso , Proteínas Adaptadoras de Sinalização CARD , Carcinoma , Linhagem Celular Tumoral , Cromatografia Líquida , Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação a DNA , Feminino , Humanos , Marcação por Isótopo , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/patologia , Poli dA-dT/farmacologia , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Adulto Jovem
17.
Mol Cells ; 27(2): 243-50, 2009 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-19277508

RESUMO

Recent studies suggest a novel role of HIF-1alpha under non-hypoxic conditions, including antibacterial and antiviral innate immune responses. However, the identity of the pathogen-associated molecular pattern which triggers HIF-1alpha activation during the antiviral response remains to be identified. Here, we demonstrate that cellular administration of double-stranded nucleic acids, the molecular mimics of viral genomes, results in the induction of HIF-1alpha protein level as well as the increase in HIF-1alpha target gene expression. Whole-genome DNA microarray analysis revealed that double-stranded nucleic acid treatment triggers induction of a number of hypoxia-inducible genes, and induction of these genes are compromised upon siRNA-mediated HIF-1alpha knock-down. Interestingly, HIF-1alpha knock-down also resulted in down-regulation of a number of genes involved in antiviral innate immune responses. Our study demonstrates that HIF-1alpha activation upon nucleic acid-triggered antiviral innate immune responses plays an important role in regulation of genes involved in not only hypoxic response, but also immune response.


Assuntos
Antivirais/metabolismo , Perfilação da Expressão Gênica , Glioblastoma/genética , Glioblastoma/imunologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Poli I-C/farmacologia , Poli dA-dT/farmacologia , Proteínas Adaptadoras de Transdução de Sinal , Biomarcadores/metabolismo , Western Blotting , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Hipóxia Celular/efeitos dos fármacos , Glioblastoma/metabolismo , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Proteínas de Ligação a RNA
18.
Mol Gen Genet ; 258(1-2): 95-103, 1998 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-9613577

RESUMO

A cis-acting element required for GCN4-independent basal-level transcription of ILV1 was previously identified in our laboratories as a binding site for the REB1 protein (Reb1p). Further deletion analysis of the ILV1 promoter region identified a second element also required for GCN4-independent basal-level ILV1 expression. This second element is an A.T-rich tract (26 As out of 32 nucleotides) situated 15 bp downstream of the Reb1p-binding site. Deletion of both the Reblp site and the poly(dA:dT) element totally eliminates basal activity of the ILV1 promoter. We show that the two elements act synergistically to control ILV1 expression and that the synergistic effect is distance dependent. We demonstrate that (i) datin (Dat1p), the only known poly (dA:dT)-binding protein in yeast, specifically binds to the ILV1 poly(dA:dT) element in vitro; (ii) Dat1p functions as a trans-activating factor in the ILV1 context; and (iii) the synergistic activation observed in vivo between the Reb1p site and the poly(dA:dT) element depends on the presence of the structural gene for Dat1p, DAT1.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Proteínas Fúngicas/genética , Regulação da Expressão Gênica , Glicoproteínas de Membrana , Proteínas de Membrana Transportadoras , Proteínas do Tecido Nervoso , Proteínas de Saccharomyces cerevisiae , Treonina Desidratase/genética , Sequência de Bases , Proteínas da Membrana Plasmática de Transporte de Dopamina , Dados de Sequência Molecular , Poli dA-dT/farmacologia , Regiões Promotoras Genéticas
19.
Anticancer Res ; 16(4A): 1843-9, 1996.
Artigo em Inglês | MEDLINE | ID: mdl-8712711

RESUMO

To gain insights into the involvement of kinases in the growth control of prostate carcinoma cells by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), immunoblot analysis and in vitro phosphorylation assays were performed using kinase discriminating effectors and extracts prepared from the androgen-dependent LNCaP and the androgen-independent JCA-1 human prostate cells. The down-regulation of PKC-alpha and -beta in JCA-1 cells was correlated with the effects of TPA. In LNCaP cells, proliferation may involve DNA-PK, proposed to act possibly via phosphorylation of the androgen receptor.


Assuntos
Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Proteínas de Ligação a DNA , Proteínas Quinases/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Linhagem Celular , Proteína Quinase Ativada por DNA , Di-Hidrotestosterona/farmacologia , Humanos , Isoenzimas/metabolismo , Cinética , Masculino , Proteínas Nucleares , Fosfoproteínas/isolamento & purificação , Fosfoproteínas/metabolismo , Fosforilação , Poli dA-dT/farmacologia , Neoplasias da Próstata , Proteína Quinase C/metabolismo , Proteína Quinase C beta , Proteína Quinase C-alfa , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Androgênicos/metabolismo , Células Tumorais Cultivadas
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