RESUMO
Pseudomonas aeruginosa (PA) is known as one kind of extracellular pathogens. However, more evidence showed that PA encounters the intracellular environment in different mammalian cell types. Little is known of innate immune factors modulating intracellular PA survival. In the present study, we proposed that interferon-ß (IFN-ß) is beneficial to the survival of PA in the cytoplasm of macrophages. Furthermore, we found that interleukin-1ß (IL-1ß) induced by PA suppresses IFN-ß response driven by the cGAS-STING-TBK1 pathway. Mechanistically, IL-1ß decreased the production of cyclic GMP-AMP (cGAMP) by activating AKT kinase. cGAMP is necessarily sufficient to stimulate the transcription of IFN-ß via the STING adaptor-TBK1 kinase-IRF3 transcription factor axis. Thus, our findings uncovered a novel module for PA intracellular survival involving IFN-ß production restricted by IL-1ß and provided a strong rationale for a potential clinical strategy against pulmonary PA infection patients. IMPORTANCE The link between innate immunity and intracellular Pseudomonas aeruginosa is unclear. Our studies illuminated the role of interferon-ß (IFN-ß) in remote intracellular PA infection. Furthermore, our experimental evidence also indicated that IL-1ß is a negative regulator of IFN-ß production and, in particular, P. aeruginosa infection. The inhibition of IFN-ß may be used as a potential therapeutic method against pulmonary PA infection.
Assuntos
Proteínas Proto-Oncogênicas c-akt , Pseudomonas aeruginosa , Animais , Humanos , Pseudomonas aeruginosa/metabolismo , Interleucina-1beta/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/metabolismo , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/metabolismo , Imunidade Inata , Mamíferos/metabolismoRESUMO
Airway microenvironment played an important role in the progression of chronic respiratory disease. Here we showed that standardized pondus hydrogenii (pH) of exhaled breath condensate (EBC) of bronchiectasis patients was significantly lower than that of controls and was significantly correlated with bronchiectasis severity index (BSI) scores and disease prognosis. EBC pH was lower in severe patients than that in mild and moderate patients. Besides, acidic microenvironment deteriorated Pseudomonas aeruginosa (P. aeruginosa) pulmonary infection in mice models. Mechanistically, acidic microenvironment increased P. aeruginosa outer membrane vesicles (PA_OMVs) released and boosted it induced the activation of interferon regulatory factor3 (IRF3)-interferonß (IFN-ß) signalling pathway, ultimately compromised the anti-bacteria immunity. Targeted knockout of IRF3 or type 1 interferon receptor (IFNAR1) alleviated lung damage and lethality of mice after P. aeruginosa infection that aggravated by acidic microenvironment. Together, these findings identified airway acidification impaired host resistance to P. aeruginosa infection by enhancing it induced the activation of IRF3-IFN-ß signalling pathway. Standardized EBC pH may be a useful biomarker of disease severity and a potential therapeutic target for the refractory P. aeruginosa infection. The study also provided one more reference parameter for drug selection and new drug discovery for bronchiectasis.
Assuntos
Bronquiectasia , Interferon Tipo I , Infecções por Pseudomonas , Animais , Concentração de Íons de Hidrogênio , Interferon beta/genética , Camundongos , Pseudomonas aeruginosa/genéticaRESUMO
Pseudomonas aeruginosa (PA) has emerged as a pressing challenge to pulmonary infection and lung damage. The LL37 peptide is an efficient antimicrobial agent against PA strains, but its application is limited because of fast clearance in vivo, biosafety concerns, and low bioavailability. Thus, an albumin-based nanodrug delivery system with reduction sensitivity was developed by forming intermolecular disulfide bonds to increase in vivo LL37 performance against PA. Cationic LL37 can be efficiently encapsulated via electrostatic interactions to exert improved antimicrobial effects. The LL37 peptide exhibits greater than 48 h of sustained released from LL37 peptide nanoparticles (LL37 PNP), and prolonged antimicrobial effects were noted as the incubation time increased. Levels of inflammatory cytokines secreted by peritoneal macrophages, including TNF-α and IL-6, were reduced significantly after LL37 PNP treatment following PA stimulation, indicating that LL37 PNP inhibits PA growth and exerts anti-inflammatory effects in vitro. In a murine model of acute PA lung infection, LL37 PNP significantly reduced TNF-α and IL-1ß expression and alleviated lung damage. The accelerated clearance of PA indicates that LL37 PNP could improve PA lung infection and the subsequent inflammation response more efficiently compared with free LL37 peptide. In conclusion, this excellent biocompatible LL37 delivery strategy may serve as an alternative approach for the application of new types of clinical treatment in future.
Assuntos
Nanopartículas , Pseudomonas aeruginosa , Albuminas , Animais , Peptídeos Catiônicos Antimicrobianos , Preparações de Ação Retardada , Pulmão , CamundongosRESUMO
The role of corticosteroids in acute lung injury (ALI) remains uncertain. This study aims to determine the underlying mechanisms of corticosteroid treatment for lipopolysaccharide (LPS)-induced inflammation and ALI. We used corticosteroid treatment for LPS-induced murine ALI model to investigate the effect of corticosteroid on ALI in vivo. Moreover, LPS-stimulated macrophages were used to explore the specific anti-inflammatory effects of corticosteroids on NLRP3-inflammasome in vitro. We found corticosteroids attenuated LPS-induced ALI, which manifested in reduction of the alveolar structure destruction, the infiltration of neutrophils and the inflammatory cytokines release of interleukin-1ß (IL-1ß) and interleukin-18 (IL-18) in Lung. In vitro, when NLRP3-inflammasome was knocked out, inflammatory response of caspase-1 activation and IL-1ß secretion was obviously declined. Further exploration, our results showed that when corticosteroid preprocessed macrophages before LPS primed, it obviously inhibited the activation of caspase-1 and the maturation of IL-1ß, which depended on inhibiting the nuclear factor-κB (NF-κB) signal pathway activation. However, when corticosteroids intervened the LPS-primed macrophages, it also negatively regulated NLRP3-inflammasome activation through suppressing mitochondrial reactive oxygen species (mtROS) production. Our results revealed that corticosteroids played a protection role in LPS-induced inflammation and ALI by suppressing both NF-κB signal pathway and mtROS-dependent NLRP3 inflammasome activation.
Assuntos
Corticosteroides/uso terapêutico , Inflamassomos/antagonistas & inibidores , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Lesão Pulmonar Aguda , Corticosteroides/farmacologia , Animais , Caspase 1/metabolismo , Dexametasona/farmacologia , Dexametasona/uso terapêutico , Ativação Enzimática/efeitos dos fármacos , Inflamassomos/metabolismo , Inflamação/induzido quimicamente , Interleucina-18/metabolismo , Lipopolissacarídeos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Modelos Biológicos , NF-kappa B/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
PM2.5, a major component of air pollutants, has caused severe health problems. It has been reported that PM2.5 index is closely associated with severity of influenza A virus (IAV) infection. However, the underlying mechanisms have not been addressed. NLRP3 inflammasome and type I interferon signaling regulate host defense against influenza infection. The present study investigated the potential effects of air pollutants on host defense against influenza infection in vitro and in vivo. In this study, different concentrations of PM2.5 were pre-exposed to macrophages and mice before IAV infection to assess the negative effects of air pollutants in virus infection. We found that exposure to PM2.5 deteriorated influenza virus infection via compromising innate immune responses manifested by a decrease IL-1ß and IFN-ß production in vitro. Meanwhile, mice exposed with PM2.5 were susceptible to PR8 virus infection due to down-regulation of IL-1ß and IFN-ß. Mechanistically, PM 2.5 exposure suppressed the NLRP3 inflammasome activation and the AHR-TIPARP signaling pathway, by which compromised the anti-influenza immunity. Thus, our study revealed that PM2.5 could alter macrophage inflammatory responses by suppressing LPS-induced activation of NLRP3 inflammasome and expression of IFN-ß during influenza infection. These findings provided us new insights in understanding that PM2.5 combining with influenza infection could enhance the severity of pneumonia.
Assuntos
Poluentes Atmosféricos/toxicidade , Inflamassomos/efeitos dos fármacos , Interferon beta/biossíntese , Proteína 3 que Contém Domínio de Pirina da Família NLR/efeitos dos fármacos , Infecções por Orthomyxoviridae/imunologia , Material Particulado/toxicidade , Animais , Inflamassomos/imunologia , Inflamassomos/metabolismo , Vírus da Influenza A Subtipo H1N1 , Interferon beta/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Infecções por Orthomyxoviridae/metabolismoRESUMO
Macrolides antibiotics have been effectively used in many chronic diseases, especially with Pseudomonas aeruginosa (P. aeruginosa) infection. The mechanisms underlying the therapeutic effects of macrolides in these diseases remain poorly understood. We established a mouse model of chronic lung infection using P. aeruginosa agar-beads, with azithromycin treatment or placebo. Lung injury, bacterial clearance, and inflammasome-related proteins were measured. In vitro, the inflammasomes activation induced by flagellin or ATP were assessed in LPS-primed macrophages with or without macrolides treatment. Plasma IL-18 levels were determined from patients who were diagnosed with bronchiectasis isolated with or without P. aeruginosa and treated with azithromycin for 3-5 days. Azithromycin treatment enhanced bacterial clearance and attenuated lung injury in mice chronically infected with P. aeruginosa, which resulted from the inhibition of caspase-1-dependent IL-1ß and IL-18 secretion. In vitro, azithromycin and erythromycin inhibited NLRC4 and NLRP3 inflammasomes activation. Plasma IL-18 levels were higher in bronchiectasis patients with P. aeruginosa isolation compared with healthy controls. Azithromycin administration markedly decreased IL-18 secretion in bronchiectasis patients. The results of this study reveal that azithromycin and erythromycin exert a novel anti-inflammatory effect by attenuating inflammasomes activation, which suggests potential treatment options for inflammasome-related diseases.
Assuntos
Bronquiectasia/tratamento farmacológico , Inflamassomos/antagonistas & inibidores , Macrolídeos/farmacologia , Infecções por Pseudomonas/prevenção & controle , Pseudomonas aeruginosa/efeitos dos fármacos , Animais , Antibacterianos/farmacologia , Azitromicina/farmacologia , Bronquiectasia/microbiologia , Células Cultivadas , Humanos , Inflamassomos/efeitos dos fármacos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Pseudomonas/microbiologiaRESUMO
CD36, a scavenger receptor, plays an important role in the progression of atherosclerosis through its interaction with oxidized low-density lipoprotein (ox-LDL). Porphyromonas gingivalis (P. gingivalis, Pg) has been shown to promote macrophage-derived foam cell formation by affecting the expression of CD36. However, the regulatory role of CD36 in macrophages infected with Pg remains largely unknown. Therefore, the aim of the present study was to explore the molecular mechanism of Pg induced CD36 expression in macrophages. Our results showed that Pg promoted ox-LDL uptake by macrophages and the formation of foam cells. Pg infection increased CD36 mRNA and protein levels in ox-LDL-untreated macrophages. Moreover, small interferon RNA (siRNA) targeting CD36 significantly reduced foam cell formation induced by Pg. Additionally, Pg stimulated nuclear translocation of p65, which directly bound to the promoters of CD36 to facilitate its transcription. Inhibition of p65, NF-κB or ERK1/2 blocked Pg-induced CD36 production; whereas, overexpression of NF-κB subunits p65 and p50 upregulated CD36. Furthermore, Ras inhibitors significantly attenuated ERK1/2 activation and CD36 expression. Taken together, the data indicated that stimulation of the ERK/NF-κB pathway by Pg led to transactivation of the CD36 promoters, thereby upregulating CD36 expression in the infected macrophages. These findings may help design new treatment strategies in atherosclerosis.
Assuntos
Infecções por Bacteroidaceae/genética , Infecções por Bacteroidaceae/microbiologia , Antígenos CD36/genética , Sistema de Sinalização das MAP Quinases , Macrófagos Peritoneais/microbiologia , NF-kappa B/metabolismo , Porphyromonas gingivalis/fisiologia , Regulação para Cima/genética , Animais , Infecções por Bacteroidaceae/patologia , Antígenos CD36/metabolismo , Feminino , Células Espumosas/metabolismo , Células Espumosas/microbiologia , Células Espumosas/patologia , Células HEK293 , Humanos , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/patologia , Camundongos , Modelos Biológicos , Regiões Promotoras Genéticas/genética , Ligação Proteica , Subunidades Proteicas/metabolismo , Células RAW 264.7RESUMO
The effect of IL-10 on macrophage migration was investigated, including the analysis of protein expression, cytokine secretion and activation of the MAPKs and NF-kB pathway. The expression of endogenic IL-10 was down-regulated in macrophage stimulated with oxLDL for indicated time. Exogenous IL-10 reversed oxLDL-inhibited chemotactic macrophage numbers by 48.18 ± 4.93% (3h), 64.8 ± 5.61% (6h) and 63.66 ± 3.05% (12h), and pretreated with SB203580 (P38 signaling inhibitor) in macrophage, oxLDL could not inhibit macrophage emigration. IL-10 significantly decreased oxLDL-mediated increase of SR-A expression, and pretreatment with ß-arrestin2 RNAi in macrophage could not change oxLDL-induced SR-A expression. IL-10 also significantly decreased oxLDL-mediated increase of ß-arrestin2 expression, and pre-treated with SR-A RNAi in macrophage, oxLDL could not induce the increase of ß-arrestin2 expression. However, IL-10 significantly reversed oxLDL-mediated inhibition of CCR7 expression about 95.7 8 ± 0.99% (mRNA) and 80.06 ± 19.46% (protein), and pretreated with P38 inhibitor SB203580 in macrophage, oxLDL could not decrease CCR7 expression. IL-10 inhibited oxLDL-mediated inhibition of MMP9 secretion about 74.02 ± 22.35%, and pretreated with P38 inhibitor SB203580 in macrophage, oxLDL could not decrease MMP9 secretion. Treatment with oxLDL increased P38-phosphorylation by 31.88 ± 2.79%, 40.24 ± 5.69% and 30.93 ± 4.66% at 15, 30 and 60 min, respectively, whereas the effect of IL-10 on the expression of phosphorylated P38 was reversed by 49.49 ± 12.12%, 70.93 ± 16.14% and 47.62 ± 6.00% in up-indicated time-points, respectively. From these data, we speculated oxLDL-SR-A-ß-arrestin2-P38-MMP9/CCR7 could play a critical role in the macrophages migration, which was blocked by IL-10 through inhibiting oxLDL uptake.
Assuntos
Movimento Celular/fisiologia , Interleucina-10/metabolismo , Lipoproteínas LDL/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Macrófagos/metabolismo , Animais , Western Blotting , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Nuclear hormone receptors respond to small molecules such as retinoids or steroids and regulate development. Signaling in the conserved p38/PMK-1 MAP kinase pathway regulates innate immunity. In this study, we show that the Caenorhabditis elegans nuclear receptor DAF-12 negatively regulates the defense against pathogens via the downstream let-7 family of microRNAs, which directly target SKN-1, a gene downstream of PMK-1. These findings identify nuclear hormone receptors as components of innate immunity that crosstalk with the p38/PMK-1 MAP kinase pathway.
Assuntos
Proteínas de Caenorhabditis elegans/imunologia , Caenorhabditis elegans/imunologia , Imunidade Inata/fisiologia , Sistema de Sinalização das MAP Quinases/imunologia , MicroRNAs/imunologia , Receptores Citoplasmáticos e Nucleares/imunologia , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Sistema de Sinalização das MAP Quinases/genética , MicroRNAs/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/imunologia , Receptores Citoplasmáticos e Nucleares/genética , Fatores de Transcrição/genética , Fatores de Transcrição/imunologiaRESUMO
The uptake of oxidized low density lipoprotein (ox-LDL) by macrophages usually leads to the formation of lipid-laden macrophages known as "foam cells," and this process plays an important role in the development of atherosclerosis. Ox-LDL activates mitogen-activated protein kinase (MAP) kinases and nuclear factor (NF)-κB, and activations of p38 and NF-κB are important for the formation of foam cells. MAP kinase phosphatase (MKP) 5 is a member of the dual specificity phosphatases (DUSPs) family that can selectively dephosphorylate activated MAPKs to regulate innate and adaptive immune responses. However, the role of MKP5 in the formation of foam cells remains unknown. Here, we found that stimulation of ox-LDL induces the expression of MKP5 in macrophages. MKP5 deficiency blocked the uptake of ox-LDL and the formation of foam cells. Further analysis revealed that deletion of MKP5 reduced the ox-LDL-induced activation of NF-κB. Also, MKP5 deficiency markedly inhibited the production of TNF-α, but enhanced the levels of TGF-ß1 in ox-LDL-stimulated macrophages. Moreover, inhibition of NF-κB by p65 RNAi significantly reduced foam cell formation in macrophages from WT mice relative to MKP5-deficient mice. Thus, MKP5 has an essential role in the formation of foam cells through activation of NF-κB, and MKP5 represents a novel target for the therapeutic intervention of atherosclerosis.
Assuntos
Fosfatases de Especificidade Dupla/imunologia , Células Espumosas/imunologia , Lipoproteínas LDL/imunologia , NF-kappa B/imunologia , Animais , Células Cultivadas , Fosfatases de Especificidade Dupla/genética , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/imunologia , Feminino , Células Espumosas/citologia , Células Espumosas/metabolismo , Regulação da Expressão Gênica , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Interferência de RNA , Receptores Depuradores/genética , Fator de Crescimento Transformador beta1/imunologia , Fator de Necrose Tumoral alfa/imunologia , Regulação para Cima , eIF-2 Quinase/genética , eIF-2 Quinase/imunologiaRESUMO
Preconditioning with sevoflurane (SPC) diminishes effusion of rat alveolar membrane during inflammation. It is not clear whether this preconditioning directly inhibits permeability of pulmonary microvascular endothelial cell (PMVEC) monolayer. In this article, we evaluated effects of SPC on permeability of PMVEC monolayer and identified signaling pathways involved in these effects. PMVEC monolayer was exposed to different conditions (5-hydroxydecanoate (5-HD), TNF-α, SPC, SPC with subsequent exposure to TNF-α and 5-HD, and SPC with subsequent exposure to TNF-α alone), and the permeability of PMVEC monolayer was assessed using FITC-bovine serum albumin (ELISA). Expression of ICAM-1 (Western blot and RT-PCR) and activation of p38 MAPK (Western blot) were also assessed. Compared to the TNF-α group, permeability of PMVEC monolayer in the SPC + TNF-α group was significantly lower. Activation of p38 MAPK was also diminished in the TNF-α group. Pre-treatment with 5-HD reverted beneficial effects of SPC. Expression of ICAM-1 was not modulated by any of the tested experimental exposures. The results of this study demonstrate that SPC is capable of diminishing the TNF-α-induced increase of permeability of PMVEC monolayer, and that this beneficial effect is partly reversed by 5-HD. Further, SPC suppresses activation of p38 MAPK.
Assuntos
Anestésicos Inalatórios/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Éteres Metílicos/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Células Cultivadas , Ácidos Decanoicos/farmacologia , Células Endoteliais/enzimologia , Endotélio Vascular/citologia , Ativação Enzimática/efeitos dos fármacos , Hidroxiácidos/farmacologia , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Ratos , Ratos Sprague-Dawley , Sevoflurano , Transdução de Sinais/efeitos dos fármacosRESUMO
Flaviviruses are a group of human pathogenic, enveloped RNA viruses that includes dengue (DENV), yellow fever (YFV), West Nile (WNV), and Japanese encephalitis (JEV) viruses. Cross-reactive antibodies against Flavivirus have been described, but most of them are generally weakly neutralizing. In this study, a novel monoclonal antibody, designated mAb 2A10G6, was determined to have broad cross-reactivity with DENV 1-4, YFV, WNV, JEV, and TBEV. Phage-display biopanning and structure modeling mapped 2A10G6 to a new epitope within the highly conserved flavivirus fusion loop peptide, the (98)DRXW(101) motif. Moreover, in vitro and in vivo experiments demonstrated that 2A10G6 potently neutralizes DENV 1-4, YFV, and WNV and confers protection from lethal challenge with DENV 1-4 and WNV in murine model. Furthermore, functional studies revealed that 2A10G6 blocks infection at a step after viral attachment. These results define a novel broadly flavivirus cross-reactive mAb with highly neutralizing activity that can be further developed as a therapeutic agent against severe flavivirus infections in humans.
Assuntos
Anticorpos Monoclonais/imunologia , Epitopos/imunologia , Infecções por Flavivirus/prevenção & controle , Flavivirus/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Reações Cruzadas/imunologia , Mapeamento de Epitopos , Infecções por Flavivirus/tratamento farmacológico , Camundongos , Internalização do VírusRESUMO
Inhibitory signaling is crucial in the regulation of the cytotoxicity of natural killer (NK) cells. Here, we show that KIR2DL1, an inhibitory receptor of NK cells, associates with supervillin, an F-actin binding protein. Interaction of supervillin with KIR2DL1 is dependent on the KIR2DL1 receptor stimulation and requires the phosphorylation of tyrosines in both ITIM motifs. "Knockdown" of expression of supervillin by RNA interference (RNAi) restores the KIR2DL1-suppressed cytotoxicity of NK cells. Inhibition of supervillin by RNAi also enhances the polarization of cytolytic granules (both granzyme B and perforin) to the synapse formed between YTS-GFP-KIR2DL1 NK cells and 721.221-HLA-Cw4 target cells. Further study reveals that supervillin is required for KIR2DL1-mediated inhibition of Vav1 and ERK phoshorylation. Moreover, we have found that binding of supervillin with KIR2DL1 facilitates the recruitment of SHPs especially SHP-2 to KIR2DL1 receptor. Thus, our findings demonstrate that supervillin is a novel molecule that associates with KIR2DL1 receptor and regulates the inhibitory signaling in NK cells.
Assuntos
Células Matadoras Naturais/fisiologia , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Receptores KIR2DL1/metabolismo , Transdução de Sinais , Motivos de Aminoácidos , Linhagem Celular Transformada , Sequência Consenso , Citotoxicidade Imunológica , Células HEK293 , Humanos , Células Matadoras Naturais/imunologia , Proteínas de Membrana/genética , Proteínas dos Microfilamentos/genética , Fosforilação , Ligação Proteica , Transporte Proteico , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Interferência de RNA , Células Tumorais CultivadasRESUMO
MAPK phosphatase-1 (MKP-1) is an archetypical member of the dual-specificity phosphatase family that deactivates MAPKs. Induction of MKP-1 has been implicated in attenuating the LPS- or peptidoglycan-induced biosynthesis of proinflammatory cytokines, but the role of noncoding RNA in the expression of the MKP-1 is still poorly understood. In this study, we show that MKP-1 is a direct target of microRNA-101 (miR-101). Transfection of miR-101 attenuates induction of MKP-1 by LPS as well as prolonged activation of p38 and JNK/stress-activated protein kinase, whereas inhibition of miR-101 enhances the expression of MKP-1 and shortens p38 and JNK activation. We also found that expression of miR-101 is induced by multiple TLR ligands, including LPS, peptidoglycan, or polyinosinic-polycytidylic acid, and that inhibition of PI3K/Akt by LY294002 or Akt RNA interference blocks the induction of miR-101 by LPS in RAW264.7 macrophage cells. Moreover, treatment of cells with dexamethasone, a widely used anti-inflammatory agent, markedly inhibits miR-101 expression and enhances the expression of MKP-1 in LPS-stimulated macrophages. Together, these results indicate that miR-101 regulates the innate immune responses of macrophages to LPS through targeting MKP-1.
Assuntos
Fosfatase 1 de Especificidade Dupla/imunologia , Imunidade Inata/fisiologia , Macrófagos/imunologia , MicroRNAs/imunologia , Animais , Anti-Inflamatórios/farmacologia , Linhagem Celular , Dexametasona/farmacologia , Fosfatase 1 de Especificidade Dupla/biossíntese , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/imunologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , MAP Quinase Quinase 4/imunologia , MAP Quinase Quinase 4/metabolismo , Macrófagos/metabolismo , Camundongos , MicroRNAs/biossíntese , Receptores Toll-Like/agonistas , Receptores Toll-Like/imunologia , Receptores Toll-Like/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
OBJECTIVE: To investigate the effect of isoflurane, sevoflurane and desflurane on the expression of ICAM-1 and VCAM-1 in LPS-induced rat lung microvascular endothelial cells (RLMVECs). METHODS: Cultured LPS-treated RLMVECs were exposed to 0.7, 1.0 or 2.0 minimum alveolar concentration (MAC) iosoflurane, sevoflurane or desflurane in 6 h. The protein expression of ICAM-1 and VCAM-1 was determined by Western blot analysis. The expression of ICAM-1 mRNA was detected by reverse-transcription polymerase chain reaction (RT-PCR). RESULT: Isoflurane at concentration of 1.0 MAC up-regulated the expression of ICAM-1 in LPS-induced RLMVECs (P <0.05); while same concentrations of sevoflurane and desflurane down-regulated the expression of ICAM-1 (P<0.05). Desflurane at concentration of 2.0 MAC up-regulated the expression of ICAM-1 in non-LPS-induced RLMVECs. All the volatile anesthetics down-regulated the expression of VCAM-1 in a dose-dependent manner. CONCLUSION: Compared to isoflurane, 1.0 MAC sevoflurane and desflurane down-regulate the expression of ICAM-1, which may be the molecule mechanism of their protective effect in acute lung injury.
Assuntos
Anestésicos Inalatórios/farmacologia , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Molécula 1 de Adesão Intercelular/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , Anestésicos Inalatórios/administração & dosagem , Animais , Células Cultivadas , Desflurano , Células Endoteliais/efeitos dos fármacos , Isoflurano/administração & dosagem , Isoflurano/análogos & derivados , Isoflurano/farmacologia , Lipopolissacarídeos/farmacologia , Pulmão/irrigação sanguínea , Éteres Metílicos/administração & dosagem , Éteres Metílicos/farmacologia , Ratos , Ratos Sprague-Dawley , SevofluranoRESUMO
Retinoic acid-inducible gene-I (RIG-I) plays an important role in antiviral response by recognizing double-stranded RNA. Here we demonstrate an unanticipated role of RIG-I in Toll-like receptor (TLR)-stimulated phagocytosis. Stimulation with lipopolysaccharide (LPS), a ligand of TLR4, induced the expression of RIG-I in macrophages. Depletion of RIG-I by RNAi or gene targeting inhibited the LPS-induced phagocytosis of bacteria. Cellular processes involved in phagocytosis, such as small GTPase Cdc42/Rac1 activation, actin polymerization, and actin-regulator Arp2/3 recruitment, were also impaired in RIG-I-deficient macrophages activated by LPS. Moreover, RIG-I(-/-) mice were found to be more susceptible to infection with Escherichia coli as compared to wild-type mice. Thus, the regulatory functions of RIG-I are strikingly broad, including a role not only in antiviral responses but in antibacterial responses as well.
Assuntos
RNA Helicases DEAD-box/imunologia , Escherichia coli/imunologia , Macrófagos/microbiologia , Fagocitose/imunologia , Receptor 4 Toll-Like/imunologia , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Animais , Linhagem Celular , Proteína DEAD-box 58 , RNA Helicases DEAD-box/genética , Infecções por Escherichia coli/imunologia , Proteínas Ativadoras de GTPase/metabolismo , Inativação Gênica , Camundongos , Camundongos Knockout , Neuropeptídeos/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Análise de Sobrevida , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTPRESUMO
The adaptor protein paxillin plays an important role in cell migration. Although the c-Jun amino-terminal kinase (JNK) phosphorylation of paxillin on Ser 178 has been found to be critical for cell migration, the precise mechanism by which JNK regulates cell migration is still not very clear. Here, the migration of human corneal epithelial (HCE) cells was used to determine which signaling pathways are involved in EGF-induced paxillin phosphorylation. Paxillin was phosphorylated on Tyr 31 and Tyr 118 after induction of migration by EGF in HCE cells. Specific inhibition of JNK activation by inhibitor SP600125 or overexpression of a dominant-negative JNK mutant not only blocked EGF-induced cell migration, but also eliminated tyrosine phosphorylation of paxillin on Tyr 31 and Tyr 118. HCE cells overexpressing paxillin-S178A mutant also exhibited lower mobility, and reduced phosphorylation of Tyr 31 and Tyr 118. However, paxillin-S178A-inhibited cell migration can be rescued by overexpression of paxillin-Y31E/Y118E mutant. Importantly, inhibition of JNK by SP600125 or overexpression of paxillin-S178A mutant prevented the association of FAK with paxillin. Taken together, these results suggest that phosphorylation of paxillin on Ser 178 by JNK is required for the association of paxillin with FAK, and subsequent tyrosine phosphorylation of paxillin.
Assuntos
Movimento Celular , Células Epiteliais/citologia , Epitélio Corneano/citologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Paxilina/metabolismo , Fosfotirosina/metabolismo , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Modelos Biológicos , Proteínas Mutantes/metabolismo , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismoRESUMO
The inhibitory signaling of natural killer (NK) cells is crucial in the regulation of innate immune responses. Here we show that the association of KIR2DL1, an inhibitory receptor of NK cells, with beta-arrestin 2 mediated recruitment of the tyrosine phosphatases SHP-1 and SHP-2 to KIR2DL1 and facilitated 'downstream' inhibitory signaling. Consequently, the cytotoxicity of NK cells was higher in beta-arrestin 2-deficient mice but was inhibited in beta-arrestin 2-transgenic mice. Moreover, beta-arrestin 2-deficient mice were less susceptible than wild-type mice to mouse cytomegalovirus infection, an effect that was abolished by depletion of NK cells. Our findings identify a previously unknown mechanism by which the inhibitory signaling in NK cells is regulated.
Assuntos
Arrestinas/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Receptores Imunológicos/imunologia , Transdução de Sinais/imunologia , Animais , Células Cultivadas , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Receptores Imunológicos/química , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , beta-Arrestina 2 , beta-ArrestinasRESUMO
RIG-I has been implicated in innate immunity by sensing intracellular viral RNAs and inducing type I IFN production. However, we have found a significant RIG-I induction in a biological setting without active viral infection-namely, during RA-induced terminal granulocytic differentiation of acute myeloid leukemias. Here, we present evidence that a significant Rig-I induction also occurs during normal myelopoiesis and that the disruption of the Rig-I gene in mice leads to the development of a progressive myeloproliferative disorder. The initiation of progressive myeloproliferative disorder is mainly due to an intrinsic defect of Rig-I(-/-) myeloid cells, which are characterized by a reduced expression of IFN consensus sequence binding protein, a major regulator of myeloid differentiation. Thus, our study reveals a critical regulatory role of Rig-I in modulating the generation and differentiation of granulocytes.
Assuntos
RNA Helicases DEAD-box/fisiologia , Regulação da Expressão Gênica , Granulócitos/citologia , Receptores do Ácido Retinoico/fisiologia , Animais , Diferenciação Celular , Proliferação de Células , Proteína DEAD-box 58 , Éxons , Granulócitos/metabolismo , Células-Tronco Hematopoéticas/citologia , Humanos , Imunidade Inata , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Células Mieloides/citologia , Transtornos Mieloproliferativos/metabolismoRESUMO
Phagocytosis and subsequent degradation of pathogens by macrophages play a pivotal role in host innate immune responses to microbial infection. Recent studies have shown that Toll-like receptors (TLRs) play an important role in promoting the clearance of bacteria by up-regulating the phagocytic activity of macrophages. However, information regarding the signaling mechanism of TLR-mediated phagocytosis is still limited. Here, we provide evidence that the stimulation of TLR4 with LPS leads to activation of multiple signaling pathways including MAP kinases, phosphatidylinositide 3-kinase (PI3K), and small GTPases in the murine macrophage-like cell line RAW264.7. Specific inhibition of Cdc42/Rac or p38 MAP kinase, but not PI3K, reduced TLR4-induced phagocytosis of bacteria. Moreover, we have found that either inhibition of actin polymerization by cytochalasin D or the knockdown of actin by RNAi markedly reduced the activation of Cdc42 and Rac by LPS. TLR4-induced activation of Cdc42 and Rac appears to be independent of MyD88. Taken together, our results described a novel actin-Cdc42/Rac pathway through which TLRs can specifically provoke phagocytosis.