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1.
PLoS Pathog ; 16(9): e1008811, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32903274

RESUMO

Damage-associated molecular patterns (DAMPs) are endogenous molecules activating the immune system upon release from injured cells. Here we show that the IFI16 protein, once freely released in the extracellular milieu of chronically inflamed tissues, can function as a DAMP either alone or upon binding to lipopolysaccharide (LPS). Specifically, using pull-down and saturation binding experiments, we show that IFI16 binds with high affinity to the lipid A moiety of LPS. Remarkably, IFI16 DAMP activity is potentiated upon binding to subtoxic concentrations of strong TLR4-activating LPS variants, as judged by TLR4-MD2/TIRAP/MyD88-dependent IL-6, IL-8 and TNF-α transcriptional activation and release in stimulated monocytes and renal cells. Consistently, using co-immunoprecipitation (co-IP) and surface plasmon resonance (SPR) approaches, we show that IFI16 is a specific TLR4-ligand and that IFI16/LPS complexes display a faster stimulation turnover on TLR4 than LPS alone. Altogether, our findings point to a novel pathomechanism of inflammation involving the formation of multiple complexes between extracellular IFI16 and subtoxic doses of LPS variants, which then signal through TLR4.


Assuntos
Inflamação/imunologia , Neoplasias Renais/imunologia , Leucemia/imunologia , Lipopolissacarídeos/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Receptor 4 Toll-Like/metabolismo , Humanos , Inflamação/metabolismo , Inflamação/patologia , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Leucemia/metabolismo , Leucemia/patologia , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais , Células Tumorais Cultivadas
2.
Life Sci ; 259: 118390, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32896556

RESUMO

AIMS: This study aimed to evaluate the function and pathway of ATP-binding cassette transporter member A1 (ABCA1)-induced anti-inflammatory response in cells at the feto-maternal interface. MAIN METHODS: The primary amniotic mesenchymal cells (AMCs), chorion cells and decidual cells were isolated from placental membranes of women with uncomplicated pregnancies at full-term (not in labor) using enzymatic digestion. Flow cytometry was used to measure the purity of isolated cells. Immunofluorescence assay was performed to detect the location of ABCA1 and toll-like receptor 4 (TLR4). Reverse transcription PCR and western blotting analyses were used to examine ABCA1, TLR4 and inflammatory factor expression in primary cells. ELISA was used to detect cytokine secretions from the primary cells. KEY FINDINGS: ABCA1 and TLR4 were mainly located in the cell nucleus and cytoplasm of feto-maternal interface cells. ABCA1 expression remained the highest in chorion cells, medium in decidual cells, and weakest in AMCs. Upregulated expression of ABCA1 decreased expression of TLR4 and the levels of pro-inflammatory factors, but increased cytoprotective factors in all cell types. In contrast, downregulated expression of ABCA1 increased the expression of TLR4 and pro-inflammatory factors, but decreased the levels of cytoprotective factors. Downregulated ABCA1 expression followed by decreased TLR4 expression using a small interference RNA (siRNA) induced reduction of interleukin (IL)-1ß and tumor necrosis factor-α (TNF-α) in all cell types. SIGNIFICANCE: ABCA1 at feto-maternal interface acts as an anti-inflammatory role by reducing the expression of TLR4 in uncomplicated pregnancies. ABCA1 might be a potential therapeutic target for preventing gestational diseases.


Assuntos
Transportador 1 de Cassete de Ligação de ATP/metabolismo , Troca Materno-Fetal , Receptor 4 Toll-Like/metabolismo , Western Blotting , Células Cultivadas , Córion/metabolismo , Decídua/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Imunofluorescência , Humanos , Microscopia Confocal , Placenta/metabolismo , Gravidez , Reação em Cadeia da Polimerase Via Transcriptase Reversa
3.
Nat Commun ; 11(1): 3816, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32732870

RESUMO

Detection of microbial components such as lipopolysaccharide (LPS) by Toll-like receptor 4 (TLR4) on macrophages induces a robust pro-inflammatory response that is dependent on metabolic reprogramming. These innate metabolic changes have been compared to aerobic glycolysis in tumour cells. However, the mechanisms by which TLR4 activation leads to mitochondrial and glycolytic reprogramming are unknown. Here we show that TLR4 activation induces a signalling cascade recruiting TRAF6 and TBK-1, while TBK-1 phosphorylates STAT3 on S727. Using a genetically engineered mouse model incapable of undergoing STAT3 Ser727 phosphorylation, we show ex vivo and in vivo that STAT3 Ser727 phosphorylation is critical for LPS-induced glycolytic reprogramming, production of the central immune response metabolite succinate and inflammatory cytokine production in a model of LPS-induced inflammation. Our study identifies non-canonical STAT3 activation as the crucial signalling intermediary for TLR4-induced glycolysis, macrophage metabolic reprogramming and inflammation.


Assuntos
Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Fator de Transcrição STAT3/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Expressão Gênica , Glicólise/efeitos dos fármacos , Inflamação/genética , Inflamação/metabolismo , Interleucina-1beta/genética , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Fator de Transcrição STAT3/genética , Serina/genética , Serina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismo , Receptor 4 Toll-Like/genética
4.
PLoS One ; 15(8): e0237034, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32745117

RESUMO

Production of IFN-γ is a key innate immune mechanism that limits replication of intracellular bacteria such as Francisella tularensis (Ft) until adaptive immune responses develop. Previously, we demonstrated that the host cell types responsible for IFN-γ production in response to murine Francisella infection include not only natural killer (NK) and T cells, but also a variety of myeloid cells. However, production of IFN-γ by mouse dendritic cells (DC) is controversial. Here, we directly demonstrated substantial production of IFN-γ by DC, as well as hybrid NK-DC, from LVS-infected wild type C57BL/6 or Rag1 knockout mice. We demonstrated that the numbers of conventional DC producing IFN-γ increased progressively over the course of 8 days of LVS infection. In contrast, the numbers of conventional NK cells producing IFN-γ, which represented about 40% of non-B/T IFN-γ-producing cells, peaked at day 4 after LVS infection and declined thereafter. This pattern was similar to that of hybrid NK-DC. To further confirm IFN-γ production by infected cells, DC and neutrophils were sorted from naïve and LVS-infected mice and analyzed for gene expression. Quantification of LVS by PCR revealed the presence of Ft DNA not only in macrophages, but also in highly purified, IFN-γ producing DC and neutrophils. Finally, production of IFN-γ by infected DC was confirmed by immunohistochemistry and confocal microscopy. Notably, IFN-γ production patterns similar to those in wild type mice were observed in cells derived from LVS-infected TLR2, TLR4, and TLR2xTLR9 knockout (KO) mice, but not from MyD88 KO mice. Taken together, these studies demonstrate the pivotal roles of DC and MyD88 in IFN-γ production and in initiating innate immune responses to this intracellular bacterium.


Assuntos
Interferon gama/metabolismo , Fator 88 de Diferenciação Mieloide/genética , Receptores Toll-Like/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Feminino , Francisella tularensis/imunologia , Imunidade Inata/imunologia , Células Matadoras Naturais/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Neutrófilos/metabolismo , Baço/metabolismo , Linfócitos T/imunologia , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Receptor Toll-Like 9/metabolismo , Receptores Toll-Like/imunologia , Tularemia/microbiologia
5.
PLoS Negl Trop Dis ; 14(8): e0008495, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32764765

RESUMO

Melioidosis is an often-severe tropical infection caused by Burkholderia pseudomallei (Bp) with high associated morbidity and mortality. Burkholderia thailandensis (Bt) is a closely related surrogate that does not require BSL-3 conditions for study. Lactoferrin is an iron-binding glycoprotein that can modulate the innate inflammatory response. Here we investigated the impact of lactoferrin on the host immune response in melioidosis. Lactoferrin concentrations were measured in plasma from patients with melioidosis and following ex vivo stimulation of blood from healthy individuals. Bt growth was quantified in liquid media in the presence of purified and recombinant human lactoferrin. Differentiated THP-1 cells and human blood monocytes were infected with Bt in the presence of purified and recombinant human lactoferrin, and bacterial intracellular replication and cytokine responses (tumor necrosis factor-α (TNF-α), interleukin-1ß and interferon-γ) were measured. In a cohort of 49 melioidosis patients, non-survivors to 28 days had significantly higher plasma lactoferrin concentrations compared to survivors (median (interquartile range (IQR)): 326 ng/ml (230-748) vs 144 ng/ml (99-277), p<0.001). In blood stimulated with heat-killed Bp, plasma lactoferrin concentration significantly increased compared to unstimulated blood (median (IQR): 424 ng/ml (349-479) vs 130 ng/ml (91-214), respectively; p<0.001). Neither purified nor recombinant human lactoferrin impaired growth of Bt in media. Lactoferrin significantly increased TNF-α production by differentiated THP-1 cells and blood monocytes after Bt infection. This phenotype was largely abrogated when Toll-like receptor 4 (TLR4) was blocked with a monoclonal antibody. In sum, lactoferrin is produced by blood cells after exposure to Bp and lactoferrin concentrations are higher in 28-day survivors in melioidosis. Lactoferrin induces proinflammatory cytokine production after Bt infection that may be TLR4 dependent.


Assuntos
Infecções por Burkholderia/metabolismo , Infecções por Burkholderia/microbiologia , Burkholderia , Lactoferrina/metabolismo , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Burkholderia pseudomallei , Células Cultivadas , Humanos , Melioidose/metabolismo , Monócitos , Receptor 4 Toll-Like/genética , Fator de Necrose Tumoral alfa/genética
6.
Int J Nanomedicine ; 15: 4125-4138, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32606668

RESUMO

Purpose: To investigate the effect and mechanism of macrophage membrane-coated nanoparticles (M-NPs) on hepatic ischemia-reperfusion injury (I/RI) caused by orthotopic liver transplantation. In addition, the advantages of TLR4+/M-NPs compared to M-NPs are discussed. Materials and Methods: We prepared biomimetic M-NPs and identified their characteristics. M-NPs were injected into an SD rat model of orthotopic liver transplantation, and the anti-inflammatory and anti-I/RI activities of M-NPs were studied in vivo and in vitro. In addition, we overexpressed macrophage membrane Toll-like receptor 4 (TLR4) in vitro and prepared TLR4+/M-NPs. Then, we assessed the characteristics and advantages of TLR4+/M-NPs. Results: The M-NPs neutralized endotoxin, inhibited the overactivation of Kupffer cells (KCs) and suppressed the secretion of inflammatory factors by inhibiting the endotoxin-mediated TLR4/MyD88/IRAK1/NF-κB signaling pathway. In an orthotopic liver transplantation model in SD rats, M-NPs showed significant therapeutic efficacy by neutralizing endotoxin and suppressing the secretion of inflammatory factors. Moreover, overexpression of TLR4 on the macrophage membrane by using a TLR4+-plasmid in vitro effectively reduced the amount of M-NPs needed to neutralize an equivalent dose of endotoxin, reducing the potential risks of NP overuse. Conclusion: This study indicates that M-NPs can effectively alleviate I/RI induced by liver transplantation.


Assuntos
Membrana Celular/metabolismo , Endotoxinas/metabolismo , Transplante de Fígado/efeitos adversos , Fígado/irrigação sanguínea , Macrófagos/metabolismo , Nanopartículas/química , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/terapia , Animais , Anti-Inflamatórios/farmacologia , Fluorescência , Lipopolissacarídeos/farmacologia , Fígado/efeitos dos fármacos , Fígado/patologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Nanopartículas/ultraestrutura , Células RAW 264.7 , Ratos Sprague-Dawley , Receptor 4 Toll-Like/metabolismo
7.
J Toxicol Sci ; 45(7): 401-409, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32612008

RESUMO

Dihydropyrazines (DHPs), including 3-hydro-2,2,5,6-tetramethylpyrazine (DHP-3), are glycation products that are spontaneously generated in vivo and ingested via food. DHPs generate various radicals and reactive oxygen species (ROS), which can induce the expression of several antioxidant genes in HepG2 cells. However, detailed information on DHP-response pathways remains elusive. To address this issue, we investigated the effects of DHP-3 on the nuclear factor-κB (NF-κB) pathway, a ROS-sensitive signaling pathway. In lipopolysaccharide-stimulated (LPS-stimulated) HepG2 cells, DHP-3 decreased phosphorylation levels of inhibitor of NF-κB (IκB) and NF-κB p65, and nuclear translocation of NF-κB p65. In addition, DHP-3 reduced the expression of Toll-like receptor 4 (TLR4) and the adaptor protein myeloid differentiation primary response gene 88 (MyD88). Moreover, DHP-3 suppressed the mRNA expression of tumor necrosis factor-alpha (TNFα), and interleukin-1 beta (IL-1ß). Taken together, these results suggest that DHP-3 acts as a negative regulator of the TLR4-MyD88-mediated NF-κB signaling pathway.


Assuntos
Dicarbetoxi-Di-Hidrocolidina/análogos & derivados , Lipopolissacarídeos/efeitos adversos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismo , Dicarbetoxi-Di-Hidrocolidina/efeitos adversos , Dicarbetoxi-Di-Hidrocolidina/toxicidade , Produtos Finais de Glicação Avançada , Células Hep G2 , Humanos , Interleucina-1beta/metabolismo , Fosforilação/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
8.
Arch Biochem Biophys ; 690: 108506, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32679197

RESUMO

A new bisepoxylignan dendranlignan A (A1) and the known compound lantibeside D (D2) was isolated from Chrysanthemum Flower, the dried capitulum of Dendranthema morifolium (Ramat.) kitam. Their structures were determined on the basis of extensive spectroscopic methods, including 1D-NMR, 2D-NMR and MS data. Additionally, A1 and D2 were evaluated for their effects on the production of inflammatory mediators in H9c2 cardiomyocytes stimulated with lipopolysaccharide (LPS). Results demonstrated that A1 and D2 decreased LPS-induced production of inflammatory cytokines TNF-α, IL-2 and IFN-γ in H9c2 cells. Both compounds also decreased the nuclear localization of c-JUN, p-P65 and p-IRF3, but did not affect the level of TLR4. Molecular docking indicated that A1 and D2 occupied the ligand binding sites of TLR4-MD2. In the present study, we for the first time discovered a new bisepoxylignan compound A1, and found that this compound has a potential to inhibit inflammation by inhibiting TLR4 signaling.


Assuntos
Chrysanthemum/química , Flores/química , Mediadores da Inflamação/metabolismo , Extratos Vegetais/química , Poli-Inos/química , Receptor 4 Toll-Like/metabolismo , Animais , Citocinas/química , Descoberta de Drogas , Humanos , Inflamação/metabolismo , Lipopolissacarídeos/química , Simulação de Acoplamento Molecular , Estrutura Molecular , Miócitos Cardíacos/metabolismo , Poli-Inos/farmacologia , Transdução de Sinais , Relação Estrutura-Atividade
9.
Expert Rev Clin Immunol ; 16(8): 751-770, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32722946

RESUMO

INTRODUCTION: Main clinical manifestations of SARS-CoV-2 infection are characterized by fever, dyspnea, and interstitial pneumonia, frequently evolving in acute respiratory distress syndrome (ARDS). AREAS COVERED: Features of coronavirus disease 2019 (COVID-19) presents some common points with interstitial lung disease (ILD) both idiopathic and related to rheumatoid arthritis (RA), typically characterized by a chronic progression over time and possibly complicated by acute exacerbation (AE). The study of common pathogenetic mechanisms, such as the involvement of toll-like receptor 4, could contribute to the knowledge and treatment of idiopathic and RA-ILD. Moreover, hyperinflammation, mainly characterized by increase of effector T-cells and inflammatory cytokines, and activation of coagulation cascade, observed in COVID-19 related ARDS have been already shown in patients with AE of idiopathic and RA-ILD. A literature search was performed in PubMed, Embase, Scopus, and Web of Science, together with a manual search in COVID-resource centers of the main journals. EXPERT OPINION: Despite the uncertainty about pathogenetic aspects about COVID-19- pneumonia, it could be a possible model for other forms of ILD and AE. The great amount of data from studies on COVID-19 could be helpful in proposing safe therapeutic approaches for RA-ILD, in understanding pathogenesis of usual interstitial pneumonia and to develop new therapeutic strategies for AE.


Assuntos
Artrite Reumatoide/patologia , Infecções por Coronavirus/patologia , Doenças Pulmonares Intersticiais/patologia , Pneumonia Viral/patologia , Artrite Reumatoide/terapia , Betacoronavirus/patogenicidade , Infecções por Coronavirus/terapia , Progressão da Doença , Humanos , Fibrose Pulmonar Idiopática/patologia , Fibrose Pulmonar Idiopática/terapia , Pulmão/patologia , Doenças Pulmonares Intersticiais/terapia , Pandemias , Pneumonia Viral/terapia , Exacerbação dos Sintomas , Receptor 4 Toll-Like/metabolismo
10.
Am J Chin Med ; 48(5): 1159-1178, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32668973

RESUMO

Hepatic ischemia-reperfusion (IR) injury remains the major cause of liver damage post-liver surgery or transplantation. Diminishing oxidative stress and inflammatory responses is a powerful channel to reduce the rate of morbidity and mortality. Gastrodin (GSTD), a bioactive compound extracted from the traditional Chinese herbal agent with a long history of clinical application in nervous system diseases, is suggested to possess anti-oxidative effects on liver diseases, such as nonalcoholic fatty liver disease. However, the therapeutic potential of GSTD in liver IR injury remains unclear. In this paper, we performed surgery to set up the 70% hepatic IR injury models in mice after a three-day pretreatment of GSTD. We found the administration of GSTD reduced liver damage, which correlated with lower histological Suzuki's score, lower serum alanine transaminase (AST) and alanine transaminase (ALT) levels, less oxidative stress, and cell apoptosis in a dose-responsive manner, as compared to the parallel control. Meanwhile, we observed a great induction of heme oxygenase-1 (HO-1) and an activation of the p38 mitogen-activated protein kinases/nuclear factor erythroid 2-related factor 2 (p38MAPK/Nrf2) pathway in response to the GSTD pretreatment, while the protective effects upon GSTD diminished in mice with HO-1 heterozygous mutation. In addition, GSTD inhibited IR induced toll-like receptor (TLR) 4, but not TLR2 in a HO-1 dependent manner, leading to a down-regulation of cytokines, such as interleukin (IL)-6 and TNF-[Formula: see text]. Collectively, our findings revealed GSTD attenuated liver IR injury via activation of the HO-1 pathway, providing a novel therapeutic strategy to minimize the IR induced oxidative stress in the process of liver transplantation.


Assuntos
Antioxidantes , Álcoois Benzílicos/administração & dosagem , Álcoois Benzílicos/farmacologia , Glucosídeos/administração & dosagem , Glucosídeos/farmacologia , Fígado , Fator 2 Relacionado a NF-E2/metabolismo , Fitoterapia , Traumatismo por Reperfusão/prevenção & controle , Transdução de Sinais/efeitos dos fármacos , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Medicamentos de Ervas Chinesas , Heme Oxigenase-1/metabolismo , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Cuidados Pré-Operatórios , Receptor 4 Toll-Like/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
11.
Arterioscler Thromb Vasc Biol ; 40(9): 2279-2292, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32611241

RESUMO

OBJECTIVE: Recruitment of neutrophils and formation of neutrophil extracellular traps (NETs) contribute to lethality in acute mesenteric infarction. To study the impact of the gut microbiota in acute mesenteric infarction, we used gnotobiotic mouse models to investigate whether gut commensals prime the reactivity of neutrophils towards formation of neutrophil extracellular traps (NETosis). Approach and Results: We applied a mesenteric ischemia-reperfusion (I/R) injury model to germ-free (GF) and colonized C57BL/6J mice. By intravital imaging, we quantified leukocyte adherence and NET formation in I/R-injured mesenteric venules. Colonization with gut microbiota or monocolonization with Escherichia coli augmented the adhesion of leukocytes, which was dependent on the TLR4 (Toll-like receptor-4)/TRIF (TIR-domain-containing adapter-inducing interferon-ß) pathway. Although neutrophil accumulation was decreased in I/R-injured venules of GF mice, NETosis following I/R injury was significantly enhanced compared with conventionally raised mice or mice colonized with the minimal microbial consortium altered Schaedler flora. Also ex vivo, neutrophils from GF and antibiotic-treated mice showed increased LPS (lipopolysaccharide)-induced NETosis. Enhanced TLR4 signaling in GF neutrophils was due to elevated TLR4 expression and augmented IRF3 (interferon regulatory factor-3) phosphorylation. Likewise, neutrophils from antibiotic-treated conventionally raised mice had increased NET formation before and after ischemia. Increased NETosis in I/R injury was abolished in conventionally raised mice deficient in the TLR adaptor TRIF. In support of the desensitizing influence of enteric LPS, treatment of GF mice with LPS via drinking water diminished LPS-induced NETosis in vitro and in the mesenteric I/R injury model. CONCLUSIONS: Collectively, our results identified that the gut microbiota suppresses NETing neutrophil hyperreactivity in mesenteric I/R injury, while ensuring immunovigilance by enhancing neutrophil recruitment.


Assuntos
Armadilhas Extracelulares/metabolismo , Microbioma Gastrointestinal , Isquemia Mesentérica/metabolismo , Mesentério/irrigação sanguínea , Infiltração de Neutrófilos , Neutrófilos/metabolismo , Traumatismo por Reperfusão/metabolismo , Vênulas/metabolismo , Animais , Bacillus subtilis/patogenicidade , Adesão Celular , Células Cultivadas , Modelos Animais de Doenças , Escherichia coli/patogenicidade , Armadilhas Extracelulares/microbiologia , Feminino , Vida Livre de Germes , Interações Hospedeiro-Patógeno , Migração e Rolagem de Leucócitos , Leucócitos/metabolismo , Leucócitos/microbiologia , Masculino , Isquemia Mesentérica/microbiologia , Isquemia Mesentérica/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Traumatismo por Reperfusão/microbiologia , Traumatismo por Reperfusão/patologia , Transdução de Sinais , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Vênulas/microbiologia , Vênulas/patologia
12.
Sci Rep ; 10(1): 10895, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32616763

RESUMO

In the past two decades, 7 coronaviruses have infected the human population, with two major outbreaks caused by SARS-CoV and MERS-CoV in the year 2002 and 2012, respectively. Currently, the entire world is facing a pandemic of another coronavirus, SARS-CoV-2, with a high fatality rate. The spike glycoprotein of SARS-CoV-2 mediates entry of virus into the host cell and is one of the most important antigenic determinants, making it a potential candidate for a vaccine. In this study, we have computationally designed a multi-epitope vaccine using spike glycoprotein of SARS-CoV-2. The overall quality of the candidate vaccine was validated in silico and Molecular Dynamics Simulation confirmed the stability of the designed vaccine. Docking studies revealed stable interactions of the vaccine with Toll-Like Receptors and MHC Receptors. The in silico cloning and codon optimization supported the proficient expression of the designed vaccine in E. coli expression system. The efficiency of the candidate vaccine to trigger an effective immune response was assessed by an in silico immune simulation. The computational analyses suggest that the designed multi-epitope vaccine is structurally stable which can induce specific immune responses and thus, can be a potential vaccine candidate against SARS-CoV-2.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Virais/imunologia , Afinidade de Anticorpos/imunologia , Betacoronavirus/química , Betacoronavirus/genética , Infecções por Coronavirus/virologia , Antígenos de Histocompatibilidade/imunologia , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Peptidil Dipeptidase A/metabolismo , Filogenia , Pneumonia Viral/virologia , Estrutura Terciária de Proteína , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Receptor 2 Toll-Like/imunologia , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/metabolismo , Vacinas Virais/metabolismo
13.
PLoS One ; 15(6): e0233643, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32479555

RESUMO

Chronic subdural hematoma (CSDH) is an angiogenic and inflammatory disease. Toll-like receptors (TLRs) transduce intracellular signals, resulting in the activation of nuclear factor κB (NF-κB), which leads to the production of inflammatory cytokines. High-mobility group box 1 (HMGB1) functions as a mediator of inflammatory responses through TLRs. In this study, we examined the expression of HMGB1 and components of the Toll-like receptor and NF-κB signaling pathways in the outer membrane of CSDH. Eight patients whose outer membrane was successfully obtained during trepanation surgery were included in this study. The expression of TLR4, myeloid differentiation factor 88 (MyD88), interleukin-1 receptor-associated kinase 4 (IRAK4), TNF receptor-associated factor 6 (TRAF6), TGFß-activated kinase 1 (Tak1), interferon regulatory factors 3 (IRF3), IκB kinase ß (IKKß), IKKγ, IκBε, IκBα, NF-κB/p65 and ß-actin was examined by Western blot analysis. The expression of TLR4, NF-κB/p65 and interleukin-6 (IL-6) was also examined by immunohistochemistry. The concentrations of HMGB1 and IL-6 in CSDH fluids were measured using ELISA kits. Above-mentioned molecules were detected in all cases. In addition, TLR4, NF-κB/p65 and IL-6 were localized in the endothelial cells of vessels within CSDH outer membranes. The concentrations of HMGB1 and IL-6 in CSDH fluids were significantly higher than that in the CSF and serum. There existed a correlation between the concentrations of HMGB1 and IL-6 in CSDH fluids. Our data suggest that HMGB1 in CSDH fluids produces the inflammatory cytokine IL-6 in endothelial cells through the Toll-like receptor and NF-κB signaling pathways. Anti-HMGB1 therapy might be a useful method to treat the growth of CSDH.


Assuntos
Proteína HMGB1/metabolismo , Hematoma Subdural Crônico/metabolismo , Interleucina-6/metabolismo , NF-kappa B/metabolismo , Receptor 4 Toll-Like/metabolismo , Idoso , Idoso de 80 Anos ou mais , Endotélio Vascular/metabolismo , Feminino , Proteína HMGB1/genética , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Quinases Associadas a Receptores de Interleucina-1/genética , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Interleucina-6/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Masculino , Pessoa de Meia-Idade , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/genética , Transdução de Sinais , Receptor 4 Toll-Like/genética
14.
J Cancer Res Clin Oncol ; 146(9): 2241-2253, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32494918

RESUMO

PURPOSE: Bone metastasis is the result of complex crosstalk between tumor cells and bone marrow cells. Bone marrow adipocytes (BMAs) are the most abundant cell type in adult bone marrow. Therefore, we explore the effects of BMAs on bone metastasis in lung cancer. METHODS: RNA-seq was used to compare the mRNA expression level of bone metastatic SBC5 cells and non-bone metastatic SBC3 cells. Rosiglitazone-induced marrow adiposity and intra-femoral injection of SBC5 cells were used to demonstrate the relationship between BMAs and SBC5 cells in vivo. Co-culture system, gene co-expression, gene ontology (GO) enrichment analysis and protein-protein interaction (PPI) network were used to explore the potential mechanism. RESULTS: BMAs specially enhance the invasion of bone metastatic SBC5 instead of non-bone metastatic SBC3 in vitro. SBC5 instead of SBC3 promoted osteoblast and osteoclast differentiation as well as de-differentiation of mature BMAs. Rosiglitazone-induced marrow adiposity significantly enhanced osteolytic lesion induced by SBC5 in vivo. RNA-seq revealed that compared with SBC3, S100A9 and S100A8 genes were the most prominent genes up-regulated in SBC5 cells. High expression of S100A8/9 in SBC5 could be responsible for the crosstalk between lung cancer cells and BMAs. More importantly, interleukin 6 receptor (IL6R), which is adjacent to S100A8/A9 in 1q21.3, was significantly up-regulated by BMAs in vitro. S100A8/A9 (1 µg/ml) could obviously enhance the osteoblastic differentiation and inhibit adipogenic differentiation, whereas TLR4 inhibitor TAK242 (10 µmol/l) significantly attenuated this effect. CONCLUSIONS: Our study suggested that bone marrow adipocyte may communicate with lung cancer cells via 1q21.3 (S100A8/A9-IL6R)-TLR4 pathway to promote osteolytic bone destruction. 1q21.3 (S100A8/A9-IL6R) is a potential target for the treatment of lung cancer bone metastasis.


Assuntos
Adipócitos/metabolismo , Medula Óssea/metabolismo , Osso e Ossos/metabolismo , Neoplasias Pulmonares/metabolismo , Osteólise/metabolismo , Receptores de Interleucina-6/metabolismo , Proteínas S100/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Células da Medula Óssea/metabolismo , Linhagem Celular Tumoral , Técnicas de Cocultura/métodos , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Osteoblastos/metabolismo , Transdução de Sinais/fisiologia , Regulação para Cima/fisiologia
15.
Life Sci ; 256: 117864, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32474021

RESUMO

As a major risk factor of acute kidney injury, renal ischemia/reperfusion (I/R) has a high mortality rate. Myeloid differentiation protein 2 (MD-2) is a secretory glycoprotein that plays an important role in inflammation. Our study aimed to explore the roles of MD-2 in I/R-induced inflammation and oxidative stress in vivo and in vitro. For the in vivo studies, male C57BL/6 mice were randomly divided into four groups: 1) sham, 2) I/R, 3) negative control for siRNA (siNC) and I/R treatment, or 4) MD-2 siRNA (siMD-2) and I/R. Levels of blood urea nitrogen and creatinine in the plasma were tested, and hematoxylin and eosin staining was performed at 24 h after I/R injury. The inflammatory cytokines TNF-α, IL-6, and MCP-1 were measured using ELISA and Real-time qPCR (RT-qPCR). Malondialdehyde (MDA) content and superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activity were estimated. For the in vitro studies, HK-2 cells were transfected with siMD-2 and then exposed to hypoxia/reoxygenation (H/R). Inflammatory cytokine expression and oxidative stress then were evaluated. We found decreased levels of blood urea nitrogen and creatinine levels after MD-2 silencing. MD-2 deficiency improved histological damage. MD-2 downregulation attenuated levels of inflammatory cytokines. Inhibition of MD-2 resulted in reduced MDA content and increased SOD, CAT, and GPx activity. Loss of function of MD-2 inhibited the H/R-induced production and expression of inflammatory cytokines. MD-2 silencing reduced MDA content after H/R, and MD-2 suppression enhanced SOD, CAT, and GPx activity. MD-2 deficiency also blocked H/R-mediated activation of the TLR4/TRAF6/NF-κB pathway, and pyrrolidinedithiocarbamate (PDTC) pretreatment strengthened the anti-inflammatory and antioxidant damage effects of MD-2 silencing. Taken together, our study revealed that MD-2 deficiency ameliorated renal I/R-induced inflammation and oxidative stress via inhibition of TLR4/TRAF6/NF-κB pathway.


Assuntos
Inflamação/patologia , Antígeno 96 de Linfócito/metabolismo , Estresse Oxidativo/genética , Traumatismo por Reperfusão/fisiopatologia , Lesão Renal Aguda/etiologia , Lesão Renal Aguda/genética , Animais , Linhagem Celular , Inativação Gênica , Humanos , Inflamação/genética , Antígeno 96 de Linfócito/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , RNA Interferente Pequeno/administração & dosagem , Fator 6 Associado a Receptor de TNF/metabolismo , Receptor 4 Toll-Like/metabolismo
16.
Life Sci ; 256: 117907, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32504751

RESUMO

Acute lung injury (ALI) and the subsequent multi-system organ failure is a serious health problem with devastating impacts on the health care systems. Indeed, the world has been facing an un-preceded situation in the past couple of months following COVID-19 infestation and the associated high-mortality rates mainly attributed to sepsis and the associated multiple organ failures of particular concern; acute respiratory distress syndrome post lung injury. The current study provides evidence on the ameliorative impact of nifuroxazide, and FDA approved antidiarrheal drug in attenuation of lipopolysaccharide (LPS)-induced ALI and myocarditis when administrated either in prophylactic or curative regimens. Nifuroxazide administration was associated with a significant improvement in lung and heart histopathological characteristics and architecture with retraction of LPS-induced inflammatory-infiltration. This was associated with retraction in serum biomarkers of cellular injury of which; LDH, CK-MB, and ALP. Nifuroxazide administration was associated with a significant improvement in both lung and heart oxidative status. Such positive outcomes were underlined by a significant inhibitory effect of nifuroxazide on lung and heart contents of toll-like receptor (4) (TLR4)/the inflammasome NALPR3/interleukin- 1ß (IL-1ß). In conclusion: Nifuroxazide attenuates LPS-induced ALI and myocardial injury via interruption of TLR4/NALPR3/IL-1ß signaling. Thus it can offer a potential approach for attenuation of sepsis in critically ill patients.


Assuntos
Lesão Pulmonar Aguda/prevenção & controle , Infecções por Coronavirus/complicações , Hidroxibenzoatos/farmacologia , Miocardite/prevenção & controle , Nitrofuranos/farmacologia , Pneumonia Viral/complicações , Sepse/tratamento farmacológico , Lesão Pulmonar Aguda/etiologia , Animais , Infecções por Coronavirus/epidemiologia , Modelos Animais de Doenças , Interleucina-1beta/metabolismo , Lipopolissacarídeos/toxicidade , Masculino , Insuficiência de Múltiplos Órgãos/etiologia , Insuficiência de Múltiplos Órgãos/prevenção & controle , Miocardite/etiologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Pandemias , Pneumonia Viral/epidemiologia , Ratos , Ratos Sprague-Dawley , Sepse/complicações , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismo
17.
Mol Immunol ; 123: 106-115, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32485469

RESUMO

Hepatocytes are the targets in autoimmune hepatitis (AIH) that results in T cell-dependent liver injury. However, hepatocytes may also affect the hepatic T cells in AIH, but the underlying mechanisms are not fully understood. Here we report that hepatocytes could secrete galectin-9 (Gal-9) to suppress the intrahepatic production of Th1 cytokine IFN-γ and restrict AIH development, but hepatocyte damage resulted in opposite effects due to release of TLR2/4 ligands that promoted the intrahepatic production of IL-1ß, IL-6, and IL-12. Through Tim-3, Gal-9 could efficiently suppress the intrahepatic T cell activation despite presence of TLR2/4 ligands, thus attenuating Th1 response in AIH. Intriguingly, intrahepatic IL-6/IL-12 suppressed the effect of TGF-ß on Treg cells. Therefore, in AIH, Gal-9 promoted Foxp3 expression and function of hepatic Treg cells through TL1A signaling, although Treg function was still impaired, compared with that in naive state. Due to its promoting effect on Treg function, together with its effect on T effector cells in a Tim-3-independent way, Gal-9 could attenuate intrahepatic IFN-γ production by hindering the increase of hepatic CD4+CD43+ T cells resulting from extrahepatic T cell activation. TLR2/4 ligands attenuated the effects of Gal-9 on Treg cells and CD4+CD43+ T cells by increasing intrahepatic IL-6 and IL-12. Blocking TLR2/4 ligands could efficiently suppress intrahepatic IFN-γ production, liver injury, and hepatic fibrosis. These findings suggest that hepatocytes paradoxically affect Th1 response in AIH due to Gal-9 expression and TLR2/4 ligands release, and that targeting TLR2/4 signaling may provide an important approach in the therapeutic strategy for AIH.


Assuntos
Galectinas/metabolismo , Hepatite Autoimune/metabolismo , Hepatócitos/fisiologia , Interferon gama/metabolismo , Fígado/metabolismo , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Células Cultivadas , Hepatite Autoimune/imunologia , Hepatite Autoimune/patologia , Ligantes , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/patologia , Células Th1/imunologia , Células Th1/metabolismo
18.
Biomed Environ Sci ; 33(5): 331-337, 2020 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-32553077

RESUMO

Objective: Hyperbaric oxygen treatment (HBOT) has demonstrated efficacy in improving hearing levels of patients with idiopathic sudden sensorineural hearing loss (ISSHL); however, the underlying mechanisms are not well understood. HBOT alleviates the inflammatory response, which is mediated by Toll-like receptor (TLR) 4 and nuclear factor (NF)-κB. In this study we investigated whether HBOT attenuates inflammation in ISHHL patients via alteration of TLR4 and NF-κB expression. Methods: ISHHL patients ( n = 120) and healthy control subjects ( n = 20) were enrolled in this study. Patients were randomly divided into medicine group treated with medicine only ( n = 60) and HBO group receiving both HBOT and medicine ( n = 60). Audiometric testing was performed pre- and post-treatment. TLR4, NF-кB, and TNF-α expression in peripheral blood of ISSHL patients and healthy control subjects was assessed by ELISA before and after treatment. Results: TLR4, NF-κB, and TNF-α levels were upregulated in ISSHL patients relative to healthy control subjects; the levels were decreased following treatment and were lower in the HBO group than that in the medicine group post-treatment ( P < 0.05 and P < 0.01). Conclusion: HBOT alleviates hearing loss in ISSHL patients by suppressing the inflammatory response induced by TLR4 and NF-κB signaling.


Assuntos
Perda Auditiva Neurossensorial/terapia , Perda Auditiva Súbita/terapia , Oxigenação Hiperbárica , Inflamação/terapia , Subunidade p50 de NF-kappa B/genética , Receptor 4 Toll-Like/genética , Adolescente , Adulto , Idoso , China , Feminino , Humanos , Inflamação/genética , Masculino , Pessoa de Meia-Idade , Subunidade p50 de NF-kappa B/metabolismo , Receptor 4 Toll-Like/metabolismo , Adulto Jovem
19.
Am J Physiol Gastrointest Liver Physiol ; 319(1): G63-G73, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32538139

RESUMO

Hyaluronic acid (HA), a glycosaminoglycan in the extracellular matrix, binds to CD44 and Toll-like receptor 4 (TLR4). We previously demonstrated that both CD44 and TLR4, but predominately TLR4, mediated HA stimulation of Lgr5+ stem cell proliferation, crypt fission, and intestinal growth in postnatal mice. Here we address the questions of which cell type expresses the relevant TLR4 in driving intestinal growth and what are the downstream events from TLR4 activation. Studies were done in 14-day-old mice: wild type (WT), mice deficient in cyclooxygenase 2 (COX2), mice deficient in myeloid cell TLR4, and mice deficient in epithelial cell epidermal growth factor receptor (EGFR). Biological end points included crypt fission and Lgr5 cell proliferation. In WT mice, treatment with NS-398 (a COX2 inhibitor), clodronate (a macrophage-depleting agent), or tyrphostin (an EGFR inhibitor) resulted in 30% reductions in crypt fission and Lgr5+ stem cell proliferation compared with control mice. Mice deficient in COX2 or myeloid TLR4 or epithelial cell EGFR all had 30% reductions in crypt fission and Lgr5+ stem cell proliferation compared with WT mice. Administration of dimethyl PGE2, a stable PGE2 analog, increased crypt fission and Lgr5+ stem cell proliferation. Administration of dimethyl PGE2 reversed the effects of NS-398, clodronate, COX2 deficiency, and myeloid TLR4 deficiency but had no effect on mice treated with tyrphostin or mice deficient in epithelial cell EGFR. We conclude that, in postnatal mice, ~30% of intestinal growth as manifested by crypt fission and Lgr5+ stem cell proliferation is driven by a novel pathway: Extracellular HA binds TLR4 on pericryptal macrophages, inducing the production of PGE2 through COX2. PGE2 transactivates EGFR in Lgr5+ epithelial stem cells, resulting in Lgr5+ stem cell proliferation and crypt fission.NEW & NOTEWORTHY This study, in newborn mice, describes a novel molecular pathway regulating Lgr5+ epithelial stem cell proliferation and normal intestinal elongation, as assessed by crypt fission. In this pathway, endogenous extracellular hyaluronic acid binds to Toll-like receptor 4 on pericryptal macrophages releasing PGE2 which binds to epidermal growth factor receptor on Lgr5+ stem cells resulting in proliferation. Lgr5+ stem cell proliferation leads to crypt fission and intestinal elongation. The demonstration that normal growth requires microbial-independent Toll-like receptor activation is novel.


Assuntos
Dinoprostona/metabolismo , Receptores ErbB/efeitos dos fármacos , Ácido Hialurônico/farmacologia , Receptor 4 Toll-Like/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Receptores ErbB/metabolismo , Ácido Hialurônico/antagonistas & inibidores , Intestinos/efeitos dos fármacos , Camundongos Knockout , Receptor 4 Toll-Like/metabolismo , Ativação Transcricional/efeitos dos fármacos
20.
Life Sci ; 256: 117935, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32526286

RESUMO

AIMS: Retinal ischemia/reperfusion (I/R) injury is common in the development of ophthalmic diseases and potentially causes blindness. In present study, the aim is to investigate the possible protective effects of puerarin on retinal I/R. MAIN METHODS: Retinal I/R injury was conducted on the left eyes of male Sprague Dawley rats, which were subsequently received treatment with puerarin. After administration, retinal I/R-induced apoptosis, oxidative stress and inflammatory responses were detected. Meanwhile, we purified retinal ganglion cells (RGCs) from 7-day-old rats. After subjected RGCs to oxygen and glucose deprivation/reoxygenation (OGD/R), apoptosis and TLR4/NLRP3 inflammasome activation in RGCs were detected. KEY FINDINGS: Puerarin prominently suppressed apoptosis, alleviated oxidative stress and suppressed TLR4/NLRP3 inflammasome activation in rats with retinal I/R injury. Consistent with our in vivo study, we found puerarin ameliorated retinal I/R injury through suppressing apoptosis and TLR4/NLRP3 inflammasome activation in RGCs. SIGNIFICANCE: Our findings reveal that puerarin plays a protective role against retinal I/R injury by alleviating RGC damage, and is beneficial for the treatment of I/R injury-caused ophthalmic diseases.


Assuntos
Isoflavonas/farmacologia , Traumatismo por Reperfusão/tratamento farmacológico , Doenças Retinianas/tratamento farmacológico , Células Ganglionares da Retina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Humanos , Inflamassomos/metabolismo , Masculino , Modelos Animais , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ratos Sprague-Dawley , Retina/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo
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