RESUMEN
Retinoic acid-inducible gene-I (RIG-I) is a cytoplasmic RNA sensor that plays an important role in innate immune responses to viral RNAs. Double-stranded RNA (dsRNA)-dependent protein kinase (PKR) is a eukaryotic initiation factor 2α (eIF2α) kinase that is initially involved in the responses of the translational machinery to dsRNA. PKR is also thought to play an essential role in antiviral innate immunity. However, the coordinated mechanisms of RIG-I and PKR that induce the expression of type I interferons (IFNs), essential cytokines involved in antiviral defense, are not completely understood. In this study, we show that PKR negatively participates in the RIG-I-mediated induction of IFN-ß expression. Stress granule (SG) formation is crucial to sequester mRNA to prevent aberrant protein synthesis by various stresses. SG formation in response to dsRNA was triggered by a PKR-mediated antiviral stress response. However, IFN-ß mRNA was not sequestered in the SGs of dsRNA-treated cells. dsRNA-induced translational silencing was thought to be PKR dependent. However, our results indicated that some proteins, including IFN-ß, were clearly translated despite PKR-mediated translational silencing. This study suggests that RIG-I responds mainly to IFN-ß expression in cells to which non-self dsRNA is introduced. In addition, PKR negatively regulates IFN-ß protein expression induced by RIG-I signaling. This may explain the essential role of PKR in fine-tuning the expression of IFN-ß in RIG-I-mediated antiviral immune responses.
Asunto(s)
ARN Bicatenario , eIF-2 Quinasa , eIF-2 Quinasa/metabolismo , Proteína 58 DEAD Box/genética , Proteína 58 DEAD Box/metabolismo , Transducción de Señal/genética , AntiviralesRESUMEN
BACKGROUND: Bronchial epithelial cells are at the front line of viral infections. Toll-like receptor 3 (TLR3) cascade causes the expression of interferon (IFN)-ß and IFN-stimulated genes (ISGs), which in turn induce an antiviral response. Members of the transmembrane protein (TMEM) family are expressed in various cell types. Although the prognostic value of TMEM2 in various cancers has been reported, its association with infectious diseases remains unknown. In this study, we investigated the effects of TMEM2 on antiviral immunity in BEAS-2B bronchial epithelial cells. METHODS AND RESULTS: TMEM2 protein was found in the cytoplasm of normal human bronchial epithelial cells and differed between organs using immunohistochemistry. Cultured BEAS-2B cells were transfected with TMEM2 siRNA, followed by administration of TLR3 ligand polyinosinic-polycytidylic acid (poly IC) or recombinant human (r(h)) IFN-ß. The expression of TMEM2, IFN-ß, ISG56, C-X-C motif chemokine ligand 10 (CXCL10) and hyaluronan were evaluated appropriately by western blotting, quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. TMEM2 expression was not altered by poly IC stimulation. Knockdown of TMEM2 increased poly IC-induced expression of IFN-ß, CXCL10, and ISG56, while IFN-ß-induced expression of ISG56 and CXCL10 were not changed by TMEM2 knockdown. The hyaluronan concentration in the medium was decreased by either TMEM2 knockdown or poly IC, but additive or synergistic effects were not observed. CONCLUSIONS: TMEM2 knockdown enhanced TLR3-mediated IFN-ß, CXCL10, and ISG56 expression in BEAS-2B cells. This implies that TMEM2 suppresses antiviral immune responses and prevents tissue injury in bronchial epithelial cells.
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Ácido Hialurónico , Receptor Toll-Like 3 , Humanos , Receptor Toll-Like 3/genética , Receptor Toll-Like 3/metabolismo , Ligandos , Poli I-C/farmacología , Células Epiteliales/metabolismo , Células Cultivadas , Quimiocina CXCL10/genéticaRESUMEN
INTRODUCTION: Invasion of viruses into the brain causes viral encephalitis, which can be fatal and causes permanent brain damage. The blood-brain barrier (BBB) protects the brain by excluding harmful substances and microbes. Brain microvascular endothelial cells are important components of the BBB; however, the mechanisms of antiviral reactions in these cells have not been fully elucidated. Zinc-finger antiviral protein (ZAP) is a molecule that restricts the infection of various viruses, and there are 2 major isoforms: ZAPL and ZAPS. Toll-like receptor 3 (TLR3), a pattern-recognition receptor against viral double-stranded RNA, is implicated in antiviral innate immune reactions. The aim of this study was to investigate the expression of ZAP in cultured hCMEC/D3 human brain microvascular endothelial cells treated with an authentic TLR3 agonist polyinosinic-polycytidylic acid (poly IC). METHODS: hCMEC/D3 cells were cultured and treated with poly IC. Expression of ZAPL and ZAPS mRNA was investigated using quantitative reverse transcription-polymerase chain reaction, and protein expression of these molecules was examined using western blotting. The role of nuclear factor-κB (NF-κB) was examined using the NF-κB inhibitor, SN50. The roles of interferon (IFN)-ß, IFN regulatory factor 3 (IRF3), tripartite motif protein 25 (TRIM25), and retinoic acid-inducible gene-I (RIG-I) in poly IC-induced ZAPS expression were examined using RNA interference. Propagation of Japanese encephalitis virus (JEV) was examined using a focus-forming assay. RESULTS: ZAPS mRNA and protein expression was upregulated by poly IC, whereas the change of ZAPL mRNA and protein levels was minimal. Knockdown of IRF3 or TRIM25 decreased the poly IC-induced upregulation of ZAPS, whereas knockdown of IFN-ß or RIG-I did not affect ZAPS upregulation. SN50 did not affect ZAPS expression. Knockdown of ZAP enhanced JEV propagation. CONCLUSION: ZAPL and ZAPS were expressed in hCMEC/D3 cells, and ZAPS expression was upregulated by poly IC. IRF3 and TRIM25 are involved in poly IC-induced upregulation of ZAPS. ZAP may contribute to antiviral reactions in brain microvascular endothelial cells and protect the brain from invading viruses such as JEV.
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Antivirales , Cerebro , Virus de la Encefalitis Japonesa (Especie) , Células Endoteliales , Microvasos , Receptor Toll-Like 3 , Humanos , Antivirales/inmunología , Antivirales/farmacología , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Células Endoteliales/inmunología , FN-kappa B/metabolismo , Poli I-C/farmacología , ARN Mensajero/metabolismo , Receptor Toll-Like 3/inmunología , Zinc , Microvasos/efectos de los fármacos , Microvasos/inmunología , Cerebro/irrigación sanguínea , Cerebro/inmunología , Virus de la Encefalitis Japonesa (Especie)/efectos de los fármacos , Virus de la Encefalitis Japonesa (Especie)/inmunologíaRESUMEN
OBJECTIVE: This paper describes the effect of Porphyromonas gingivalis (P gingivalis) lipopolysaccharide (LPS) on the expression of interleukin-6 (IL-6) and C-C motif chemokine ligand 2 (CCL2) in cultured hCMEC/D3 human brain microvascular endothelial cells. BACKGROUND: P gingivalis is one of the important pathogens in periodontitis, and periodontitis is a risk factor for brain disorders including cerebrovascular diseases and Alzheimer's disease. However, the mechanisms underlying the pathogenesis of P gingivalis-mediated brain diseases are incompletely understood. Effects of P gingivalis LPS on brain endothelial cells are not known well. METHODS: The hCMEC/D3 human brain microvascular endothelial cells were cultured and treated with P gingivalis LPS. The expression of IL-6 and CCL2 mRNA and protein was examined using quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Effect of inhibitors of Toll-like receptor (TLR) 2, TLR4, nuclear factor-κB (NF-κB), p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) was also investigated. Phosphorylation of NF-κB p65, p38 MAPK and JNK was examined using Western blotting. RESULTS: P gingivalis LPS-induced mRNA and protein expression of IL-6 and CCL2 in hCMEC/D3 cells in a concentration-dependent manner at the concentration of 0.5-50 µg/mL. Maximal mRNA expression of IL-6 and CCL2 was found 2 and 4 hours after stimulation, respectively. Induction of IL-6 and CCL2 by P gingivalis LPS was almost completely inhibited by pretreatment of cells with TLR4 inhibitor but not by TLR2 inhibitor. Treatment of cells with P gingivalis LPS for up to 2 hours induced phosphorylation of NF-κB p65, p38 MAPK and JNK. IL-6 induction was decreased by pretreatment of cells with NF-κB inhibitor SN50 or p38 MAPK inhibitor SB203580, while CCL2 induction was reduced by SN50 or JNK inhibitor SP600125. CONCLUSIONS: IL-6 and CCL2 produced upon P gingivalis LPS stimulation may contribute to the inflammatory reactions in brain endothelial cells and subsequent neurological disorders such as cerebrovascular and Alzheimer's diseases.
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Infecciones por Bacteroidaceae/metabolismo , Encéfalo/citología , Quimiocina CCL2/metabolismo , Interleucina-6/metabolismo , Lipopolisacáridos , Porphyromonas gingivalis , Infecciones por Bacteroidaceae/inmunología , Células Cultivadas , Quimiocinas/metabolismo , Células Endoteliales/metabolismo , Humanos , Ligandos , FN-kappa B/metabolismo , Periodontitis/complicaciones , ARN Mensajero/genética , Receptor Toll-Like 4/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Since aortic valve stenosis (AVS) is the most frequent and serious valvular heart disease in the elderly, and is accompanied by irreversible valve calcification, medicinal prevention of AVS is important. Although we recently demonstrated that human aortic valve interstitial cells (HAVICs) obtained from patients with AVS were highly sensitive to ectopic calcification stimulation, the cell types contributing to calcification are unknown. We aimed to immunocytochemically characterize HAVICs and identify their contribution to valve calcification. HAVICs were isolated from patients with AVS and cultured on non-coated dishes. Immunocytochemical features and HAVIC differentiation were analyzed in passage 1 (P1). The immunohistochemical features of the calcified aortic valve were analyzed. Most cultured P1 HAVICs were CD73-, CD90-, and CD105-positive, and CD45-and CD34-negative. HAVICs were vascular endothelial growth factor receptor 2 (VEGFR2)-positive; however, approximately half were α-smooth muscle actin (SMA)-positive, colonized, and easily differentiated into osteoblastic cells. Calcified aortic valve immunohistochemistry showed that all cells were positive for VEGFR2 and partly α-SMA. Further, VEGFR2-positive cells were more sensitive to tumor necrosis factor-α-induced ectopic calcification with or without α-SMA positivity. We conclude that HAVICs obtained from patients with AVS are VEGFR2-positive undifferentiated mesenchymal cells and may contribute to aortic valve ectopic calcification.
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Estenosis de la Válvula Aórtica/metabolismo , Estenosis de la Válvula Aórtica/patología , Válvula Aórtica/citología , Válvula Aórtica/metabolismo , Válvula Aórtica/patología , Calcinosis/metabolismo , Calcinosis/patología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Actinas/metabolismo , Anciano , Estenosis de la Válvula Aórtica/etiología , Calcinosis/etiología , Células Cultivadas , Femenino , Humanos , Masculino , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
C-X-C motif chemokine 10 (CXCL10) is an inflammatory chemokine and a key molecule in the pathogenesis of rheumatoid arthritis (RA). Melanoma differentiation-associated gene 5 (MDA5) is an RNA helicase that plays a role in innate immune and inflammatory reactions. The details of the regulatory mechanisms of CXCL10 production and the precise role of MDA5 in RA synovitis have not been fully elucidated. The aim of this study was to examine the role of MDA5 in regulating CXCL10 expression in cultured human rheumatoid fibroblast-like synoviocytes (RFLS). RFLS was stimulated with Toll-like receptor 3 (TLR3) ligand polyinosinic:polycytidylic acid (poly I:C), a synthetic double-stranded RNA mimetic. Expression of interferon beta (IFN-ß), MDA5, and CXCL10 was measured by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), western blotting, and enzyme-linked immunosorbent assay. A neutralizing antibody of IFN-ß and siRNA-mediated MDA5 knockdown were used to determine the role of these molecules in regulating CXCL10 expression downstream of TLR3 signaling in RFLS. Poly I:C induced IFN-ß, MDA5, and CXCL10 expression in a concentration- and time-dependent manner. IFN-ß neutralizing antibody suppressed the expression of MDA5 and CXCL10, and knockdown of MDA5 decreased a part of CXCL10 expression (p < 0.001). The TLR3/IFN-ß/CXCL10 axis may play a crucial role in the inflammatory responses in RA synovium, and MDA5 may be partially involved in this axis.
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Artritis Reumatoide/genética , Quimiocina CXCL10/genética , Helicasa Inducida por Interferón IFIH1/genética , Receptor Toll-Like 3/genética , Artritis Reumatoide/patología , Biomimética , Fibroblastos/metabolismo , Fibroblastos/patología , Regulación de la Expresión Génica/genética , Humanos , Inmunidad Innata/genética , Inflamación/genética , Inflamación/patología , Interferón beta/genética , ARN Bicatenario/genética , ARN Bicatenario/farmacología , Transducción de Señal/genética , Membrana Sinovial/metabolismo , Membrana Sinovial/patología , Sinoviocitos/metabolismo , Sinoviocitos/patologíaRESUMEN
BACKGROUND: Although toll-like receptor 3 (TLR3) signaling is involved in the development of certain chronic kidney diseases, the specific molecular mechanisms underlying inflammatory reactions via activation of TLR3 signaling in human podocytes remain unclear. Interleukin (IL)-6 is a pleiotropic cytokine associated with innate and adaptive immune responses; however, little is known about the implication of IL-6 via the activation of regional TLR3 signaling in the inflammatory reactions in human podocytes. METHODS: We treated immortalized human podocytes with polyinosinic-polycytidylic acid (poly IC), an authentic viral double-stranded RNA, and assessed the expression of IL-6, monocyte chemoattractant protein-1 (MCP-1), and C-C motif chemokine ligand 5 (CCL5) using quantitative real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. To further elucidate the poly IC-induced signaling pathway, we subjected the cells to RNA interference against IFN-ß and IL-6. RESULTS: We found that the activation of TLR3 induced expression of IL-6, MCP-1, CCL5, and IFN-ß in human podocytes. RNA interference experiments revealed that IFN-ß was involved in the poly IC-induced expression of IL-6, MCP-1, and CCL5. Interestingly, IL-6 knockdown markedly increased the poly IC-induced expression of MCP-1 and CCL5. Further, treatment of cells with IL-6 attenuated the expression of CCL5 and MCP-1 mRNA and proteins. CONCLUSION: IL-6 induced by TLR3 signaling negatively regulates the expression of representative TLR3 signaling-dependent proinflammatory chemokines in human podocytes.
Asunto(s)
Quimiocinas/metabolismo , Inflamación/metabolismo , Interleucina-6/metabolismo , Podocitos/metabolismo , Transducción de Señal/inmunología , Receptor Toll-Like 3/metabolismo , HumanosRESUMEN
INTRODUCTION: Various viruses including a novel coronavirus (SARS-CoV-2) can infect the kidney. When viruses invade the glomeruli from the bloodstream, glomerular endothelial cells (GECs) initiate the innate immune reactions. We investigated the expression of interferon (IFN)-induced protein with tetratricopeptide repeats (IFIT) 1/2/3, antiviral molecules, in human GECs treated with a toll-like receptor (TLR) 3 agonist. Role of IFIT1/2/3 in the expression of C-X-C motif chemokine ligand 10 (CXCL10) was also examined. METHODS: Human GECs were cultured and stimulated with polyinosinic-polycytidylic acid (poly IC), a synthetic TLR3 agonist. Real-time qPCR, Western blotting, and ELISA were used to examine the expression of IFIT1/2/3, IFN-ß, and CXCL10. RNA interference against IFN-ß or IFIT1/2/3 was also performed. RESULTS: Expression of IFIT1/2/3 and CXCL10 was induced by poly IC in GECs. The inductions were inhibited by RNA interfering of IFN-ß. Knockdown of IFIT1/2/3 decreased the CXCL10 expression. Knockdown of IFIT3 decreased the expression of IFIT1 and IFIT2 proteins. CONCLUSION: IFIT1/2/3 and CXCL10 were induced by poly IC via IFN-ß in GECs. IFIT1/2/3 may increase the expression of CXCL10 which induces lymphocyte chemotaxis and may inhibit the replication of infected viruses. These molecules may play a role in GEC innate immune reactions in response to viruses.
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Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Proteínas Reguladoras de la Apoptosis/biosíntesis , Quimiocina CXCL10/biosíntesis , Péptidos y Proteínas de Señalización Intracelular/biosíntesis , Glomérulos Renales/metabolismo , Proteínas de Unión al ARN/biosíntesis , Receptor Toll-Like 3/agonistas , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Reguladoras de la Apoptosis/genética , Células Cultivadas , Quimiocina CXCL10/genética , Relación Dosis-Respuesta a Droga , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Glomérulos Renales/citología , Glomérulos Renales/efectos de los fármacos , Poli I-C/farmacología , Proteínas de Unión al ARN/genética , Receptor Toll-Like 3/metabolismoRESUMEN
BACKGROUND: Glomerular endothelial cells (GECs) are directly exposed to circulating viral particles in the glomerulus. Although viral infections may trigger the development of acute kidney injury or the worsening of pre-existing chronic kidney disease, the specific molecular mechanisms underlying antiviral reactions via the activation of endothelial Toll-like receptor 3 signaling in the kidney remain to be determined. Interferon (IFN)-induced transmembrane protein 1 (IFITM1), a member of interferon-stimulated gene protein family, is involved in the prevention of viral entry into cerebral vascular endothelial cells, respiratory epithelial cells, and endometrium. However, as far as we are aware, the implication of IFITM1 associated with viral infections in GECs has not been investigated to date. METHODS: Cultured, normal human GECs were treated with polyinosinic-polycytidylic acid (poly IC), a synthesized viral double-stranded RNA, then the expression of IFITM1 was examined by quantitative real-time reverse transcription-polymerase chain reaction and western blotting. To further elucidate the poly IC-induced signaling pathway, the cells were applied to RNA interference against IFN-ß, nuclear factor-κB p65, and IFN regulatory factor 3. We also conducted an immunofluorescence study to examine endothelial IFITM1 expression in biopsy specimens from patients with chronic kidney disease. RESULTS: We found that the activation of Toll-like receptor 3 induced endothelial expression of IFITM1, and that this involved IFN regulatory factor 3 and IFN-ß, but not nuclear factor-κB. Intense endothelial IFITM1 immunoreactivity was observed in biopsy specimens from patients with lupus nephritis. CONCLUSIONS: Antiviral reaction-related endothelial expression of IFITM1 may be involved, at least in part, in the development of particularly in lupus nephritis. Further detailed studies of the implication of interferon stimulated genes, including IFITM1 in GECs are needed.
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Antígenos de Diferenciación/genética , Células Endoteliales , Glomérulos Renales/citología , Poli I-C , Células Cultivadas , Humanos , Factor 3 Regulador del Interferón , Interferón beta , Receptor Toll-Like 3 , Factor de Transcripción ReIARESUMEN
OBJECTIVE: Although anti-malarial agents, chloroquine (CQ) and hydroxychloroquine (HCQ) are currently used for the treatment of systemic lupus erythematosus, their efficacy for lupus nephritis (LN) remains unclear. Given that upregulation of glomerular Toll-like receptor 3 (TLR3) signaling plays a pivotal role in the pathogenesis of LN, we examined whether CQ and HCQ affect the expression of the TLR3 signaling-induced representative proinflammatory chemokines, monocyte chemoattractant protein-1 (MCP-1), and C-C motif chemokine ligand 5 (CCL5) in cultured human glomerular endothelial cells (GECs). METHODS: We examined the effect of polyinosinic-polycytidylic acid (poly IC), an agonist of TLR3, on MCP-1, CCL5 and interferon (IFN)-ß expression in GECs. We then analyzed whether pretreatment with CQ, HCQ, or dexamethasone (DEX) inhibits poly IC-induced expression of these chemokines using real-time quantitative reverse transcriptase PCR and ELISA. Phosphorylation of signal transducers and activator of transcription protein 1 (STAT1) was examined using western blotting. RESULTS: Poly IC increased MCP-1 and CCL5 expression in a time- and concentration-dependent manner in GECs. Pretreating cells with CQ, but not DEX, attenuated poly IC-induced MCP-1 and CCL5 expression; however, HCQ pretreatment attenuated poly IC-induced CCL5, but not MCP-1. HCQ did not affect the expression of IFN-ß and phosphorylation of STAT-1. CONCLUSION: Considering that TLR3 signaling is implicated, at least in part, in LN pathogenesis, our results suggest that anti-malarial agents exert a protective effect against the development of inflammation in GECs, as postulated in LN. Interestingly, CQ is a rather powerful inhibitor compared with HCQ on TLR3 signaling-induced chemokine expression in GECs. In turn, these findings may further support the theory that the use of HCQ is safer than CQ in a clinical setting. However, further detailed studies are needed to confirm our preliminary findings.
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Antimaláricos/farmacología , Quimiocina CCL5/metabolismo , Quimiocinas/genética , Células Endoteliales/metabolismo , Receptor Toll-Like 3/metabolismo , Línea Celular , Células Cultivadas , Quimiocina CCL5/genética , Cloroquina/farmacología , Humanos , Inflamación/metabolismo , Interferón beta/metabolismo , Glomérulos Renales/citología , Nefritis Lúpica/tratamiento farmacológico , Poli I-C/metabolismo , Poli I-C/farmacología , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 3/genéticaRESUMEN
BACKGROUND: Propolis is a natural product collected by worker bees from a variety of plant species. As a type of propolis, Brazilian green propolis contains a large amount of artepillin C. Artepillin C is a cinnamic acid derivative and has been shown to have a wide variety of biological functions, including anti-inflammatory, antiviral and antitumor activities, in both cell culture and animal models. However, how propolis is digested and absorbed remains to be elucidated. Moreover, blood artepillin C levels after propolis intake have not been shown in human studies. RESULTS: A randomized, single-blind placebo-controlled study on the effect of Brazilian green propolis on serum artepillin C levels was conducted with healthy volunteers. The participants (n = 133) were randomly allocated in an approximately 2:1 ratio to two groups: propolis (n = 91) and placebo (n = 42). The participants took daily propolis or placebo, and blood tests were performed on day 0 (before propolis intake) and days 1, 3 and 7. Artepillin C was detected in serum in almost all individuals in the propolis groups. No serum artepillin C was detected in the placebo group. Serum artepillin C levels in the female group tended to be higher than those in the male group. In the female group, menstrual status was unrelated to serum artepillin C levels. CONCLUSION: These results suggested that propolis intake might be more effective for females than for males. © 2021 Society of Chemical Industry.
Asunto(s)
Fenilpropionatos/sangre , Própolis/metabolismo , Adulto , Anciano , Animales , Antiinflamatorios/sangre , Abejas , Brasil , Femenino , Humanos , Masculino , Persona de Mediana Edad , Própolis/análisis , Adulto JovenRESUMEN
OBJECTIVE: Brain microvascular endothelial cells are integral components of the blood-brain barrier and play a role in protecting the brain from invading microbes. CXC motif chemokine ligand 1 (CXCL1) induces the chemotaxis of neutrophils, and neutrophils are important in host defense in the brain. However, dysregulated neutrophil infiltration leads to brain diseases. Toll-like receptor 3 (TLR3) is a pattern recognition receptor that recognizes viral double-stranded RNA (dsRNA). The aim of this study was to investigate the effect of an TLR3 agonist on the expression of CXCL1 in brain vascular endothelial cells. METHODS: hCMEC/D3 human cerebral microvascular endothelial cells were cultured and treated with polyinosinic-polycytidylic acid (poly IC), a potent synthetic dsRNA agonist for TLR3. The production of CXCL1 mRNA and protein was assessed by real-time RT-PCR and ELISA. The expression of CXCL1 was compared with that of CXCL8. The effect of pretreatment of cells with a NF-κB inhibitor (SN50), a p38 mitogen-activated protein kinase (MAPK) inhibitor (SB203580), a c-Jun N-terminal kinase (JNK) inhibitor (SP600125), an interferon (IFN) regulatory factor 3 inhibitor (MRT67307), and an anti-type I IFN-neutralizing antibody mixture was examined. Phosphorylation of p38 was examined using Western blotting. RESULTS: Treating cultured hCMEC/D3 human cells with poly IC induced the expression of CXCL1 as well as another chemokine CXCL8. Pretreatment of cells with SN50, SB203580, and SP600125 decreased the induction of CXCL1 by poly IC. However, it was not affected by MRT67307 or by an anti-type I IFN-neutralizing antibody mixture. Pretreatment of cells with SN50 decreased the poly IC-induced phosphorylation of p38. CONCLUSIONS: Poly IC induces the expression of CXCL1 in hCMEC/D3 cells. NF-κB, p38 MAPK, and JNK are involved in this reaction. There is a cross-talk between NF-κB and p38, and NF-κB partially regulates phosphorylation of p38. CXCL1 produced by brain microvascular endothelial cells may contribute to the brain's defense against viral infection and various neurological diseases associated with neutrophil accumulation.
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Barrera Hematoencefálica/metabolismo , Quimiocina CXCL1/biosíntesis , Células Endoteliales/metabolismo , Infiltración Neutrófila/fisiología , Poli I-C/farmacología , Barrera Hematoencefálica/efectos de los fármacos , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Humanos , Transducción de Señal/fisiologíaRESUMEN
The recognition of nonself dsRNA by retinoic acid-inducible gene-I (RIG-I) leads to the engagement of RIG-I-like receptor signaling. In addition, nonself dsRNA triggers a robust latent RNase (RNase L) activation and leads to the degradation of ribosomal structures and cell death. In contrast, nonself ssRNA is known to be recognized by TLR 7/8 in immune cells such as plasmacytoid dendritic cells and B cells, but little is known regarding the involvement of nonself ssRNA in antiviral signaling in nonimmune cells, including epithelial cells. Moreover, the fate of intracellular nonself ssRNA remains unknown. To address this issue, we developed a quantitative RT-PCR-based approach that monitors the kinetics of nonself ssRNA cleavage following the transfection of HeLa human cervical carcinoma cells, using model nonself ssRNA. We discovered that the degradation of ssRNA is independent of RIG-I and type I IFN signaling because ssRNA did not trigger RIG-I-mediated antiviral signaling. We also found that the kinetics of self (5'-capped) and nonself ssRNA decay were unaltered, suggesting that nonself ssRNA is not recognized by nonimmune cells. We further demonstrated that the cleavage of nonself ssRNA is accelerated when nonself dsRNA is also introduced into cells. In addition, the cleavage of nonself ssRNA is completely abolished by knockdown of RNase L. Overall, our data demonstrate the important role of dsRNA-RNase L in nonself ssRNA degradation and may partly explain the positive regulation of the antiviral responses in nonimmune cells.
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Antivirales/metabolismo , ARN Bicatenario/metabolismo , ARN Viral/metabolismo , Transducción de Señal/fisiología , Línea Celular Tumoral , Proteína 58 DEAD Box/inmunología , Proteína 58 DEAD Box/metabolismo , Endorribonucleasas/metabolismo , Células HeLa , Humanos , ARN Bicatenario/inmunología , ARN Viral/inmunología , Transducción de Señal/inmunología , Receptores Toll-Like/inmunología , Receptores Toll-Like/metabolismoRESUMEN
Purpose and aim of the study: Bronchial epithelial cells play an important role in immune response against viral infections. Toll-like receptor 3 (TLR3) is a pathogen recognition receptor that recognizes viral double-stranded RNA (dsRNA). Activation of TLR3 induces the expression of interferon (IFN)-ß, and newly synthesized IFN-ß exhibits anti-viral activity by upregulating the expression of IFN-stimulated genes (ISGs). ISG56 encodes a multifunctional protein with tetratricopeptide motifs and is involved in anti-viral reactions through various mechanisms. Expression of chemokines such as CXCL10, which induces leukocyte chemotaxis, is essential for defense against airway microbes. However, regulation of chemokine expression by ISG56 in bronchial epithelial cells has not been fully investigated. The aim of this study was to examine the expression of ISG56 and its role in CXCL10 production in BEAS-2B bronchial epithelial cells treated with dsRNA.Materials and methods: BEAS-2B bronchial epithelial cells were treated with polyinosinic-polycytidylic acid (poly IC), a synthetic TLR3 ligand. The mRNA and protein expression levels of ISG 56 were analyzed by quantitative reverse transcription polymerase chain reaction and western blotting. The effect of knocking down TLR3, IFN-ß, and ISG56 was examined using RNA interference. The protein expression of CXCL10 in culture medium was measured using an enzyme-linked immunosorbent assay.Results: Poly IC induced ISG56 expression in a concentration- and time- dependent manner. RNA interference showed that ISG56 induction was inhibited by knockdown of TLR3 or IFN-ß and that ISG 56 knockdown decreased CXCL10 expression.Conclusions: ISG56 was induced by poly IC through TLR3/IFN-ß axis, and ISG56 may positively regulated CXCL10 expression in BEAS-2B cells. ISG56 may modulate anti-viral innate immunity, at least in part, by regulating the expression of CXCL10 in bronchial epithelial cells.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Quimiocina CXCL10/metabolismo , Células Epiteliales/metabolismo , Proteínas de Unión al ARN/metabolismo , Transducción de Señal/fisiología , Receptor Toll-Like 3/metabolismo , Regulación hacia Arriba/fisiología , Células Cultivadas , Células Epiteliales/efectos de los fármacos , Humanos , Inmunidad Innata/efectos de los fármacos , Inmunidad Innata/fisiología , Interferón beta/metabolismo , Poli I-C/farmacología , Interferencia de ARN/fisiología , ARN Bicatenario/metabolismo , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Background: Endothelial expression of membrane-bound fractalkine/CX3CL1 (Fkn) reportedly acts as a strong mediator of inflammation. Toll-like receptor 3 (TLR3) axes are thought to play some roles in the development of chronic glomerulonephritis (CGN) including lupus nephritis (LN). However, detailed mechanism of TLR3-mediated Fkn expression in glomerular endothelial cells (GECs) remains to be elucidated.Methods: We examined the effect of polyinosinic-polycytidylic acid (poly IC) on Fkn expression in cultured human GECs. Fkn mRNA and protein levels were quantified by real-time PCR and enzyme-linked immunosorbent assay, respectively. To further elucidate the effects of poly IC on this signaling pathway, we used small-interfering RNA (siRNA) to knockdown expression of TLR3, nuclear factor (NF)-κB p65, interferon (IFN)-ß, and IFN regulatory factor 3 (IRF3). We then analyzed whether pretreatment of chloroquine or dexamethasone (DEX) inhibits poly IC-induced Fkn expression.Results: We found that poly IC-induced Fkn expression in GECs, and that this involved NF-κB, IFN-ß, and IRF3. Pretreating cells with chloroquine, but not DEX attenuated poly IC-induced Fkn expression in GECs.Conclusion: Since the activation of TLR3/NF-κB/IFN-ß/Fkn and TLR3/IRF3/Fkn axes is involved in inflammatory reactions in GECs, intervention of glomerular TLR3 signaling may be a suitable therapeutic strategy for treating CGN especially LN.
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Quimiocina CX3CL1/metabolismo , Células Endoteliales/metabolismo , Transducción de Señal , Receptor Toll-Like 3/metabolismo , Células Cultivadas , Quimiocina CX3CL1/genética , Cloroquina/farmacología , Células Endoteliales/efectos de los fármacos , Humanos , Interferón beta/metabolismo , Glomérulos Renales/citología , Glomérulos Renales/metabolismo , FN-kappa B/metabolismo , Poli I-C/farmacologíaRESUMEN
BACKGROUND/AIMS: Dysregulation of interleukin-6 (IL-6) production in residual renal cells may play a pivotal role in the development of glomerulonephritis (GN). Given that Toll-like receptor 3 (TLR3) signaling has been implicated in the pathogenesis of some forms of GN, we examined activated TLR3-mediated IL-6 signaling in cultured normal human glomerular endothelial cells (GECs). METHODS: We treated GECs with polyinosinic-polycytidylic acid (poly IC), an authentic double-stranded RNA, and analyzed the expression of IL-6 and the cytosolic viral RNA sensors retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation associated gene 5 (MDA5) using reverse transcription quantitative real-time polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assays. To further elucidate the effects of poly IC on this signaling pathway, we subjected the cells to small interfering RNA (siRNA) against TLR3, interferon (IFN)-ß, RIG-I, and MDA5. RESULTS: We found that poly IC induced the expression of RIG-I, MDA5 and IL-6 via TLR3/IFN-ß signaling in GECs. siRNA experiments revealed that both MDA5 and RIG-I were involved in the poly IC-induced expression of IL-6, with MDA5 being upstream of RIG-I. CONCLUSION: Interestingly, cytosolic sensors of viral RNA were found to be involved in IL-6 production via TLR3 signaling in GECs. Regional activation of TLR3/IFN-ß/ MDA5/RIG-I/IL-6 axis due to viral and "pseudoviral" infections is involved in innate immunity and inflammatory reactions in GECs. We believe this signaling pathway also plays a pivotal role in the development of some forms of GN.
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Interleucina-6/biosíntesis , Glomérulos Renales/citología , Receptor Toll-Like 3/metabolismo , Células Cultivadas , Proteína 58 DEAD Box/metabolismo , Células Endoteliales/metabolismo , Glomerulonefritis/etiología , Humanos , Inflamación , Helicasa Inducida por Interferón IFIH1/metabolismo , Poli I-C/farmacología , ARN Viral , Receptores Inmunológicos , Transducción de SeñalRESUMEN
Recent investigations have clarified the importance of mitochondria in various age-related degenerative diseases, including late-onset Alzheimer's disease and Parkinson's disease. Although mitochondrial disturbances can be involved in every step of disease progression, several observations have demonstrated that a subtle mitochondrial functional disturbance is observed preceding the actual appearance of pathophysiological alterations and can be the target of early therapeutic intervention. The signals from damaged mitochondria are transferred to the nucleus, leading to the altered expression of nuclear-encoded genes, which includes mitochondrial proteins (i.e., mitochondrial retrograde signaling). Mitochondrial retrograde signaling improves mitochondrial perturbation (i.e., mitohormesis) and is considered a homeostatic stress response against intrinsic (ex. aging or pathological mutations) and extrinsic (ex. chemicals and pathogens) stimuli. There are several branches of the mitochondrial retrograde signaling, including mitochondrial unfolded protein response (UPRMT), but recent observations increasingly show the importance of the ISR-ATF4 pathway in mitochondrial retrograde signaling. Furthermore, Nrf2, a master regulator of the oxidative stress response, interacts with ATF4 and cooperatively upregulates a battery of antioxidant and antiapoptotic genes while repressing the ATF4-mediated proapoptotic gene, CHOP. In this review article, we summarized the upstream and downstream mechanisms of ATF4 activation during mitochondrial stresses and disturbances and discuss therapeutic intervention against degenerative diseases by using Nrf2 activators.
RESUMEN
BACKGROUND/AIMS: Cylindromatosis (CYLD), a deubiquitinase, negatively regulates nuclear factor-κB in various cells. However, its potential roles in glomerular inflammation remain unclear. Because the activation of the Toll-like receptor 3 (TLR3)/type I interferon (IFN) pathways plays a pivotal role in chronic kidney diseases (CKD), we examined the role of CYLD in the TLR3 signaling in cultured human mesangial cells (MCs). METHODS: We stimulated CYLD-silenced MCs with polyinosinic-polycytidylic acid (poly IC), a synthetic analogue of dsRNA, and studied representative TLR3/IFN-ß pathways (i.e., TLR3/IFN-ß/retinoic acid-inducible gene-I (RIG-I)/CCL5, and TLR3/IFN-ß/melanoma differentiation associated gene 5 (MDA5)/CXCL10 axes) using RT-PCR, western blotting, and ELISA. We also used immunofluorescence staining and microscopy to examine mesangial CYLD expression in biopsied specimens from patients with CKD. RESULTS: CYLD silencing resulted in an increase of poly IC-induced RIG-I and MDA5 protein levels and increased CCL5 and CXCL10 mRNA and protein expression, but unexpectedly decreased mRNA expressions of RIG-I and MDA5. Interestingly, CYLD silencing did not affect IFN-ß or the phosphorylated STAT1 (signal transducers and activator of transcription protein 1). CYLD was highly expressed in biopsied specimens from patients with proliferative lupus nephritis (LN). CONCLUSION: CYLD inhibits post-transcriptional regulation of RIG-I and MDA5 expression following TLR3 activation in MCs. CYLD may be involved in the pathogenesis of CKD, especially pathogenesis of LN.
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Enzima Desubiquitinante CYLD/fisiología , Inflamación , Células Mesangiales/metabolismo , Transducción de Señal , Receptor Toll-Like 3/metabolismo , Células Cultivadas , Proteína 58 DEAD Box/metabolismo , Humanos , Helicasa Inducida por Interferón IFIH1/metabolismo , Nefritis Lúpica , Receptores Inmunológicos , Insuficiencia Renal Crónica/etiologíaRESUMEN
BACKGROUND: Chloroquine has been reported to protect against renal damage in lupus nephritis (LN); however, its detailed mechanism in glomerular inflammation remains unclear. Upregulation of the type-I interferon (IFN) system plays a pivotal role in LN pathogenesis, therefore, we examined whether chloroquine inhibits toll-like receptor 3 (TLR3)/IFN-ß signaling in cultured normal human mesangial cells (MCs). METHODS: We examined chloroquine effect on the representative TLR3/IFN-ß-signaling axis, TLR3/IFN-ß/retinoic acid-inducible gene-I (RIG-I)/CCL5 in MCs treated with polyinosinic-polycytidylic acid (poly IC), a synthetic viral dsRNA analog and analyzed the expression of these molecules using reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA). Furthermore, we subjected MCs to RNA interference against NF-κB p65. RESULTS: Pretreatment of cells with chloroquine attenuated IFN-ß, RIG-I and CCL5 expression and phosphorylation of STAT1 induced by poly IC, but not IFN-ß-induced phosphorylation of STAT1 and RIG-I expression induced by IFN-ß. Knockdown of p65 inhibited the poly IC-induced IFN-ß expression, and chloroquine pretreatment decreased the nuclear poly IC-induced translocation of NF-κB p65 in MCs. CONCLUSION: These results suggest that chloroquine attenuates mesangial TLR3 signaling in the early phase of NF-κB activation. Considering that TLRs/type-I IFNs signaling is implicated in LN pathogenesis, our results may further support regional renoprotective effects of chloroquine in treating LN.
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Antirreumáticos/farmacología , Cloroquina/farmacología , Interferón beta/metabolismo , Nefritis Lúpica/metabolismo , Células Mesangiales/metabolismo , Transducción de Señal , Receptor Toll-Like 3/metabolismo , Células Cultivadas , Humanos , Interferón beta/genética , Células Mesangiales/efectos de los fármacos , FN-kappa B/genética , FN-kappa B/metabolismo , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo , Receptor Toll-Like 3/genéticaRESUMEN
BACKGROUND/AIMS: Activation of Toll-like receptor 3 (TLR3) signaling followed by type I interferon (IFN) expression is crucial in antiviral and "pseudoviral" immune reactions in renal mesangial cells (MCs). These reactions are probably involved in the pathogenesis of chronic kidney disease (CKD). However, the role of IFN-induced 35-kDa protein 35 (IFI35), a type I IFN-dependent transcript, in glomerular inflammation is unclear. Here, we aimed to investigate the expression and the role of IFI35 in IFN-ß/retinoic acid-inducible gene-I (RIG-I)/CCL5 and IFN-ß/melanoma differentiation-associated gene 5 (MDA5)/CXCL10 axes in MCs. METHODS: We treated human MCs with polyinosinic-polycytidylic acid (poly IC), an authentic double-stranded RNA, then analysed the IFI35 expression by reverse transcription-polymerase chain reaction and western blotting. To examine the regulation of IFI35 expression, we subjected MCs to RNA interference (siRNA) against IFN-ß, RIG-I, and MDA5. RESULTS: Activation of TLR3 by poly IC induces the IFI35 expression in MCs. siRNA against IFN-ß inhibited poly IC-induced IFI35 expression. Knockdown of IFI35 resulted in a decrease of poly IC-induced RIG-I and MDA5 protein as well as decreased CCL5 and CXCL10 mRNA and protein expression. However, it did not affect the expression of none of phosphorylated signal transducers or activator of transcription (STAT) 1 protein, or RIG-I and MDA5 in mRNA levels. CONCLUSION: Regional expression of IFI35 and its dysregulation may be involved in the pathogenesis of glomerular inflammation in CKD.