RESUMO
BACKGROUND: Heart failure with preserved ejection fraction (HFpEF) is associated with systemic inflammation, obesity, metabolic syndrome, and gut microbiome changes. Increased trimethylamine-N-oxide (TMAO) levels are predictive for mortality in HFpEF. The TMAO precursor trimethylamine (TMA) is synthesized by the intestinal microbiome, crosses the intestinal barrier and is metabolized to TMAO by hepatic flavin-containing monooxygenases (FMO). The intricate interactions of microbiome alterations and TMAO in relation to HFpEF manifestation and progression are analyzed here. METHODS: Healthy lean (L-ZSF1, n = 12) and obese ZSF1 rats with HFpEF (O-ZSF1, n = 12) were studied. HFpEF was confirmed by transthoracic echocardiography, invasive hemodynamic measurements, and detection of N-terminal pro-brain natriuretic peptide (NT-proBNP). TMAO, carnitine, symmetric dimethylarginine (SDMA), and amino acids were measured using mass-spectrometry. The intestinal epithelial barrier was analyzed by immunohistochemistry, in-vitro impedance measurements and determination of plasma lipopolysaccharide via ELISA. Hepatic FMO3 quantity was determined by Western blot. The fecal microbiome at the age of 8, 13 and 20 weeks was assessed using 16s rRNA amplicon sequencing. RESULTS: Increased levels of TMAO (+ 54%), carnitine (+ 46%) and the cardiac stress marker NT-proBNP (+ 25%) as well as a pronounced amino acid imbalance were observed in obese rats with HFpEF. SDMA levels in O-ZSF1 were comparable to L-ZSF1, indicating stable kidney function. Anatomy and zonula occludens protein density in the intestinal epithelium remained unchanged, but both impedance measurements and increased levels of LPS indicated an impaired epithelial barrier function. FMO3 was decreased (- 20%) in the enlarged, but histologically normal livers of O-ZSF1. Alpha diversity, as indicated by the Shannon diversity index, was comparable at 8 weeks of age, but decreased by 13 weeks of age, when HFpEF manifests in O-ZSF1. Bray-Curtis dissimilarity (Beta-Diversity) was shown to be effective in differentiating L-ZSF1 from O-ZSF1 at 20 weeks of age. Members of the microbial families Lactobacillaceae, Ruminococcaceae, Erysipelotrichaceae and Lachnospiraceae were significantly differentially abundant in O-ZSF1 and L-ZSF1 rats. CONCLUSIONS: In the ZSF1 HFpEF rat model, increased dietary intake is associated with alterations in gut microbiome composition and bacterial metabolites, an impaired intestinal barrier, and changes in pro-inflammatory and health-predictive metabolic profiles. HFpEF as well as its most common comorbidities obesity and metabolic syndrome and the alterations described here evolve in parallel and are likely to be interrelated and mutually reinforcing. Dietary adaption may have a positive impact on all entities.
Assuntos
Modelos Animais de Doenças , Progressão da Doença , Microbioma Gastrointestinal , Insuficiência Cardíaca , Metilaminas , Volume Sistólico , Função Ventricular Esquerda , Animais , Insuficiência Cardíaca/fisiopatologia , Insuficiência Cardíaca/microbiologia , Insuficiência Cardíaca/metabolismo , Metilaminas/metabolismo , Metilaminas/sangue , Masculino , Obesidade/microbiologia , Obesidade/fisiopatologia , Obesidade/metabolismo , Oxigenases/metabolismo , Oxigenases/genética , Fígado/metabolismo , Biomarcadores/sangue , Fezes/microbiologia , Ratos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Bactérias/metabolismo , DisbioseRESUMO
Introduction: The bacterial protein toxin Pasteurella multocida toxin (PMT) mediates RANKL-independent osteoclast differentiation. Although these osteoclasts are smaller, their resorptive activity is high which helps in efficient destruction of nasal turbinate bones of pigs. Methods: The proteome of bone marrow-derived macrophages differentiated into osteoclasts with either RANKL or PMT was analysed. The results were verified by characterizing the metabolic activity using Seahorse analysis, a protein translation assay, immunoblots, real-time PCR as well as flow cytometry-based monitoring of mitochondrial activity and ROS production. A Gαq overexpression system using ER-Hoxb8 cells was used to identify Gαq-mediated metabolic effects on osteoclast differentiation and function. Results: PMT induces the upregulation of metabolic pathways, which included strong glycolytic activity, increased expression of GLUT1 and upregulation of the mTOR pathway. As OxPhos components were expressed more efficiently, cells also displayed increased mitochondrial respiration. The heterotrimeric G protein Gαq plays a central role in this hypermetabolic cell activation as it triggers mitochondrial relocalisation of pSerSTAT3 and an increase in OPA1 expression. This seems to be caused by a direct interaction between STAT3 and OPA1 resulting in enhanced mitochondrial respiration. Overexpression of Gαq mimicked the hypermetabolic phenotype observed for PMT-induced osteoclasts and resulted in higher glycolytic and mitochondrial activity as well as increased bone resorptive activity. In addition, rheumatoid arthritis (RA) patients showed an increase in GNAQ expression, especially in the synovial fluid. Discussion: Our study suggests that Gαq plays a key role in PMT-induced osteoclastogenesis. Enhanced expression of GNAQ at the site of inflammation in RA patients indicates its pathophysiological relevance in the context of inflammatory bone disorders.
Assuntos
Osteoclastos , Pasteurella multocida , Animais , Suínos , Osteoclastos/metabolismo , Macrófagos/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/farmacologia , Diferenciação Celular/fisiologia , Metabolismo Energético , Ligante RANK/metabolismoRESUMO
Antigen-presenting cells (APCs) regulate the balance of our immune response toward microbes. Whereas immunogenic APCs boost inflammation and activate lymphocytes, the highly plastic cells can switch into a tolerogenic/suppressive phenotype that dampens and resolves the response. Thereby the initially mediated inflammation seems to prime the switch of APCs while the strength of activation determines the grade of the suppressive phenotype. Recently, we showed that pathogen recognition receptor-mediated pro-inflammatory cytokines reprogram differentiating human blood monocytes in vitro toward an immunosuppressive phenotype through prolonged activation of signal transducer and activator of transcription (STAT) 3. The TLR7/8 ligand R848 (Resiquimod) triggers the high release of cytokines from GM-CSF/IL-4-treated monocytes. These cytokines subsequently upregulate T cell suppressive factors, such as programmed death-ligand 1 (PD-L1) and indolamin-2,3-dioxygenase (IDO) through cytokine receptor-mediated STAT3 activation. Here, we reveal an essential role for the microRNA (miR, miRNA) hsa-miR-99b/let-7e/miR-125a cluster in stabilizing the suppressive phenotype of R848-stimulated APCs on different levels. On the one hand, the miR cluster boosts R848-stimulated cytokine production through regulation of MAPkinase inhibitor Tribbles pseudokinase 2, thereby enhancing cytokine-stimulated activation of STAT3. One the other hand, the STAT3 inhibitor suppressor of cytokine signaling-1 is targeted by the miR cluster, stabilizing the STAT3-induced expression of immunosuppressive factors PD-L1 and IDO. Finally, hsa-miR-99b/let-7e/miR-125a cluster regulates generation of the suppressive tryptophan (Trp) metabolite kynurenine by targeting the tryptophanyl-tRNA synthetase WARS, the direct competitor of IDO in terms of availability of Trp. In summary, our results reveal the hsa-miR-99b/let-7e/miR-125a cluster as an important player in the concerted combination of mechanisms that stabilizes STAT3 activity and thus regulate R848-stimulated suppressive APCs.
Assuntos
Células Apresentadoras de Antígenos/metabolismo , Interações Hospedeiro-Patógeno/genética , MicroRNAs/genética , Células Apresentadoras de Antígenos/efeitos dos fármacos , Células Apresentadoras de Antígenos/imunologia , Biomarcadores , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Citocinas/biossíntese , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imidazóis/farmacologia , Imunomodulação/efeitos dos fármacos , Imunomodulação/genética , Imunofenotipagem , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/metabolismo , Família Multigênica , Interferência de RNA , Fator de Transcrição STAT3/genéticaRESUMO
During infection pathogen-associated molecular patterns activate immune cells to initiate a cascade of reactions leading to inflammation and the activation of the adaptive immune response culminating in the elimination of foreign pathogens. However, shortly after activation of the host defense machinery, a return to homeostasis is preferred to prevent inflammation-induced tissue damage. This switch from the initial immunogenic to the subsequent tolerogenic phase after clearance of the infection can be mediated through highly plastic peripheral monocytes. Our studies reveal that an early encounter with toll-like receptor 7/8-ligand R848 mediates a strong pro-inflammatory monocytic phenotype that primes its own reprogramming toward an immunosuppressive one. Previously, we showed that these R848-treated antigen-presenting cells (APCs) fail to activate allogeneic T cells and induce regulatory T cells (Tregs) through signal transducer and activator of transcription 3 (STAT3)-dependent PD-L1. Here, we further demonstrate that R848-treated APCs suppress CD3/CD28-mediated and dendritic cell-mediated T cell activation and that adenosine and indoleamine 2,3-dioxygenase/kynurenin pathways are involved in tolerance induction. Reprogramming of monocytes after R848 stimulation requires the pro-inflammatory cytokine IL-1ß and a boosted IL-6 release. The subsequent autocrine prolonged activation of STAT3 induces direct upregulation of tolerogenic factors which finally downregulate proliferation of activated T cells and mediate Tregs. Thereby our study suggests that inflammatory cytokines, such as IL-1ß and IL-6, should be considered as mediators of resolution of inflammation.
RESUMO
INTRODUCTION: Hyporesponsiveness of human lamina propria immune cells to microbial and nutritional antigens represents one important feature of intestinal homeostasis. It is at least partially mediated by low expression of the innate response receptors CD11b, CD14, CD16 as well as the cystine-glutamate transporter xCT on these cells. Milieu-specific mechanisms leading to the down-regulation of these receptors on circulating monocytes, the precursor cells of resident macrophages, are mostly unknown. METHODS: Here, we addressed the question whether the short chain fatty acid n-butyrate, a fermentation product of the mammalian gut microbiota exhibiting histone deacetylase inhibitory activity, is able to modulate expression of these receptors in human circulating monocytes. RESULTS: Exposure to n-butyrate resulted in the downregulation of CD11b, CD14, as well as CD16 surface expression on circulating monocytes. XCT transcript levels in circulating monocytes were also reduced following exposure to n-butyrate. Importantly, treatment resulted in the downregulation of protein and gene expression of the transcription factor PU.1, which was shown to be at least partially required for the expression of CD16 in circulating monocytes. PU.1 expression in resident macrophages in situ was observed to be substantially lower in healthy when compared to inflamed colonic mucosa. CONCLUSIONS: In summary, the intestinal microbiota may support symbiosis with the human host organism by n-butyrate mediated downregulation of protein and gene expression of innate response receptors as well as xCT on circulating monocytes following recruitment to the lamina propria. Downregulation of CD16 gene expression may at least partially be caused at the transcriptional level by the n-butyrate mediated decrease in expression of the transcription factor PU.1 in circulating monocytes.
Assuntos
Butiratos/imunologia , Imunidade Inata , Monócitos/imunologia , Monócitos/metabolismo , Receptores Imunológicos/metabolismo , Adulto , Sistemas de Transporte de Aminoácidos Acídicos/genética , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Antígenos de Bactérias/imunologia , Biomarcadores , Regulação para Baixo , Exposição Ambiental , Feminino , Imunofluorescência , Regulação da Expressão Gênica , Humanos , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas/metabolismo , Receptores Imunológicos/genética , Transativadores/metabolismoRESUMO
Bone and joint infections (BJIs) are often difficult to treat. Staphylococcus spp. is the major pathogen causing these infections, which is often associated with biofilm formation on prosthetic materials. Therapeutic measures are complex, ranging from surgical intervention to initial intravenous and supportive long-term oral antibiotic therapy. The options for oral antimicrobial therapy are limited, mainly due to the resistance profile of the causative pathogen and the unfavourable pharmacodynamic and pharmacokinetic properties of most antibiotics in biofilm. Data analysis over a 5-year period was performed on staphylococci isolated from BJI patients in the Orthopaedic Department of the University Hospital Heidelberg (Heidelberg, Germany) to assess the plausibility of fusidic acid (FA)-based alternative oral treatment regimens. Six percent of BJIs were caused by meticillin-resistant Staphylococcus aureus (MRSA), and multiresistance was common. Over 75% of MRSA in BJIs were resistant to the commonly used rifampicin (RIF)-based combinations. Resistance to FA-based combinations was high. However, over 80% were susceptible to the combination RIF+FA. In coagulase-negative staphylococci, resistance to RIF-based combinations was similar to FA-based combinations. Almost two-thirds of the isolates tested were susceptible to RIF+FA. These data suggest FA as a possible option as a substitution for RIF or as a combination companion in case of resistance or unavailability.
Assuntos
Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Farmacorresistência Bacteriana , Ácido Fusídico/farmacologia , Ácido Fusídico/uso terapêutico , Osteoartrite/microbiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus/efeitos dos fármacos , Administração Oral , Antibacterianos/administração & dosagem , Alemanha , Hospitais Universitários , Humanos , Staphylococcus/isolamento & purificaçãoRESUMO
Histone deacetylase (HDAC) inhibitors (HDACi) are clinically approved anticancer drugs that have important immune-modulatory properties. We report the surprising finding that HDACi promote LPS-induced IL-1ß processing and secretion in human and murine dendritic cells and murine macrophages. HDACi/LPS-induced IL-1ß maturation and secretion kinetics differed completely from those observed upon inflammasome activation. Moreover, this pathway of IL-1ß secretion was dependent on caspase-8 but was independent of the inflammasome components NACHT, LRR, and PYD domains-containing protein 3, apoptosis-associated speck-like protein containing a carboxyl-terminal caspase-recruitment domain, and caspase-1. Genetic studies excluded HDAC6 and HDAC10 as relevant HDAC targets in this pathway, whereas pharmacological inhibitor studies implicated the involvement of HDAC11. Treatment of mice with HDACi in a dextran sodium sulfate-induced colitis model resulted in a strong increase in intestinal IL-1ß, confirming that this pathway is also operative in vivo. Thus, in addition to the conventional inflammasome-dependent IL-1ß cleavage pathway, dendritic cells and macrophages are capable of generating, secreting, and processing bioactive IL-1ß by a novel, caspase-8-dependent mechanism. Given the widespread interest in the therapeutic targeting of IL-1ß, as well as the use of HDACi for anti-inflammatory applications, these findings have substantial clinical implications.
Assuntos
Caspase 8/imunologia , Células Dendríticas/imunologia , Inibidores de Histona Desacetilases/farmacologia , Interleucina-1beta/metabolismo , Macrófagos/imunologia , Animais , Células da Medula Óssea , Proteínas de Transporte , Caspase 1/genética , Caspase 1/imunologia , Inibidores de Caspase/farmacologia , Caspases/genética , Caspases Iniciadoras , Células Cultivadas , Colite/induzido quimicamente , Sulfato de Dextrana , Histona Desacetilases/imunologia , Inflamassomos/imunologia , Lipopolissacarídeos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLRRESUMO
Bacterial components are recognized by the immune system through activation of the inflammasome, eventually causing processing of the proinflammatory cytokine interleukin-1? (IL-1?), a pleiotropic cytokine and one of the most important mediators of inflammation, through the protease caspase-1. Synthesis of the precursor protein and processing into its bioactive form are tightly regulated, given that disturbed control of IL-1? release can cause severe autoinflammatory diseases or contribute to cancer development. We show that the bacterial Pasteurella multocida toxin (PMT) triggers Il1b gene transcription in macrophages independently of Toll-like receptor signaling through RhoA/Rho-kinase-mediated NF-?? activation. Furthermore, PMT mediates signal transducer and activator of transcription (STAT) protein-controlled granzyme A (a serine protease) expression in macrophages. The exocytosed granzyme A enters target cells and mediates IL-1? maturation independently of caspase-1 and without inducing cytotoxicity. These findings show that macrophages can induce an IL-1?-initiated immune response independently of inflammasome activity.
Assuntos
Granzimas/metabolismo , Inflamassomos/metabolismo , Interleucina-1beta/biossíntese , Transdução de Sinais , Animais , Apoptose/efeitos dos fármacos , Proteínas de Bactérias/farmacologia , Toxinas Bacterianas/farmacologia , Caspase 1/metabolismo , Humanos , Inflamassomos/efeitos dos fármacos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Janus Quinases/metabolismo , Camundongos , NF-kappa B/metabolismo , Perforina/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptores Toll-Like/metabolismo , Transcrição Gênica/efeitos dos fármacos , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
The significance of bacterial RNA recognition for initiating innate immune responses against invading pathogens has only recently started to be elucidated. Bacterial RNA is an important trigger of inflammasome activation, resulting in caspase-1-dependent cleavage of pro-IL-1ß into the active form. It was reported previously that prolonged treatment with IFN-γ can inhibit IL-1ß production at the level of both transcription and Nlrp3 inflammasome activation in an NO-dependent manner. As a result of the delayed kinetics of NO generation after IFN-γ stimulation, these effects were only observed at later time points. We report that IFN-γ suppressed bacterial RNA and LPS induced IL-1ß transcription in primary murine macrophages and dendritic cells by an additional, very rapid mechanism that was independent of NO. Costimulation with IFN-γ selectively attenuated binding of NF-κB p65 to the IL-1ß promoter, thus representing a novel mechanism of IL-1ß inhibition by IFN-γ. Transcriptional silencing was specific for IL-1ß because expression of other proinflammatory cytokines, such as TNF, IL-6, and IL-12p40, was not affected. Furthermore, by suppressing IL-1ß production, IFN-γ impaired differentiation of Th17 cells and production of neutrophil chemotactic factor CXCL1 in vitro. The findings provide evidence for a rapid immune-modulating effect of IFN-γ independent of NO.
Assuntos
Imunidade Inata/imunologia , Interferon gama/metabolismo , Interleucina-1beta/biossíntese , NF-kappa B/metabolismo , Regiões Promotoras Genéticas , Animais , Western Blotting , Diferenciação Celular/imunologia , Imunoprecipitação da Cromatina , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/genética , Óxido Nítrico/metabolismo , RNA Bacteriano/imunologia , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Th17/citologia , Células Th17/imunologiaRESUMO
IRAK4, a serine/threonine kinase is a central adaptor protein in TLR signaling. To better understand the clinical significance of IRAK4 deficiency we examined the impact of IRAK4 on bacterial recognition in human monocytes. We show that IRAK4 knockdown modulates monocyte-derived cytokine secretion in response to Staphylococcus aureus and Streptococcus pneumoniae, resulting in decreased IL-12 and elevated IL-10 production, a finding also reproducible with ligands for TLR2 and TLR4. In contrast, silencing of MyD88 leads to a complete loss of cytokine secretion, indicating that IRAK4 acts as a differential regulator of bacteria/TLR-induced cytokine secretion downstream of MyD88. Further analysis revealed that this modulatory function results from IRAK4-mediated suppression of protein kinase B (PKB/Akt). Release of suppression upon IRAK4 silencing (but not MyD88 knockdown) increases phosphorylation of PKB/Akt, counteracts NF-κB activation and finally results in a monocyte phenotype with tolerogenic features, thus unleashing Akt- and mTOR-dependent release of IL-10, along with concomitant phosphorylation of FOXO transcription factors. In line with these observations IRAK4-deficient monocytes failed to induce allogeneic CD8(+) and CD4(+) T-cell responses, an effect reverted by neutralization of IL-10. Taken together, our data highlight an unexpected role of IRAK4, Akt, and mTOR in the regulation of tolerance in human monocytes.
Assuntos
Quinases Associadas a Receptores de Interleucina-1/imunologia , Monócitos/imunologia , Fator 88 de Diferenciação Mieloide/metabolismo , Infecções Pneumocócicas/imunologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/imunologia , Streptococcus pneumoniae/imunologia , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Células Cultivadas , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/metabolismo , Humanos , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Ativação Linfocitária/genética , Monócitos/microbiologia , Fator 88 de Diferenciação Mieloide/genética , NF-kappa B/metabolismo , Proteína Oncogênica v-akt/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/genética , Serina-Treonina Quinases TOR/imunologiaRESUMO
Sepsis is a major cause of mortality in hospitalized patients worldwide, with lethality rates ranging from 30 to 70â%. Sepsis is caused by a variety of different pathogens, and rapid diagnosis is of outstanding importance, as early and adequate antimicrobial therapy correlates with positive clinical outcome. In recent years, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) fingerprinting has become a powerful tool in microbiological diagnostics. The direct identification of micro-organisms in a positive blood culture by MALDI-TOF MS can shorten the diagnostic procedure significantly. Therefore, the aim of the present study was to evaluate whether identification rates could be improved by using the new Sepsityper kit from Bruker Daltonics for direct isolation and identification of bacteria from positive blood cultures by MALDI-TOF MS compared with the use of conventional separator gel columns, and to integrate the MALDI-TOF MS-based identification method into the routine course of blood culture diagnostics in the setting of a microbiological laboratory at a university hospital in Germany. The identification of Gram-negative bacteria by MALDI-TOF MS was significantly better using the Sepsityper kit compared with a separator gel tube-based method (99 and 68â% correct identification, respectively). For Gram-positive bacteria, only 73â% were correctly identified by MALDI-TOF with the Sepsityper kit and 59â% with the separator gel tube assay. A major problem of both methods was the poor identification of Gram-positive grape-like clustered cocci. As differentiation of Staphylococcus aureus from coagulase-negative staphylococci is of clinical importance, a PCR was additionally established that was capable of identifying S. aureus directly from positive blood cultures, thus closing this diagnostic gap. Another benefit of the PCR approach is the possibility of directly detecting the genes responsible for meticillin resistance in staphylococci and for vancomycin resistance in enterococci, which is of high importance for early adequate treatment. Both of the described methods were finally integrated into a protocol for fast and effective identification of bacteria from positive blood cultures.
Assuntos
Bactérias/classificação , Bactérias/isolamento & purificação , Técnicas Bacteriológicas/métodos , Sangue/microbiologia , Reação em Cadeia da Polimerase/métodos , Sepse/diagnóstico , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Bactérias/química , Alemanha , Humanos , Sensibilidade e Especificidade , Sepse/microbiologiaRESUMO
In LPS-treated macrophages, activation of STAT3 is considered to be crucial for terminating the production of inflammatory cytokines. By analyzing the role of MAPK-activated protein kinase (MK) 2 and MK3 for LPS-induced STAT3 activation in macrophages, the present study provides evidence that MK2 is crucial for STAT3 activation in response to LPS because it prevents MK3 from impeding IFNß gene expression. Accordingly, LPS-induced IFNß gene expression is down-regulated in MK2-deficient macrophages and can be reconstituted by additional ablation of the MK3 gene in MK2/3(-/-) macrophages. This is in contrast to LPS-induced IL-10 expression, which essentially requires the presence of MK2. Further analysis of downstream signaling events involved in the transcriptional regulation of IFNß gene expression suggests that, in the absence of MK2, MK3 impairs interferon regulatory factor 3 protein expression and activation and inhibits nuclear translocation of p65. This inhibition of p65 nuclear translocation coincides with enhanced expression and delayed degradation of IκBß, whereas expression of IκBα mRNA and protein is impaired in the absence of MK2. The observation that siRNA directed against IκBß is able to reconstitute IκBα expression in MK2(-/-) macrophages suggests that enhanced expression and delayed degradation of IκBß and impaired NFκB-dependent IκBα expression are functionally linked. In summary, evidence is provided that MK2 regulates LPS-induced IFNß expression and downstream STAT3 activation as it restrains MK3 from mediating negative regulatory effects on NFκB- and interferon regulatory factor 3-dependent LPS signaling.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/fisiologia , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/biossíntese , Interferon beta/genética , Interleucina-10/biossíntese , Interleucina-10/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Macrófagos/citologia , Camundongos , Camundongos Knockout , Proteínas Serina-Treonina Quinases/genética , Fator de Transcrição STAT3/genética , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismoRESUMO
In this issue of Blood, Roger and colleagues present data on the magnitude of influence that broad-spectrum HDAC inhibitors exert on TLR-driven immune responses, thus demonstrating that HDAC inhibitors are immunosuppressive drugs.
RESUMO
Airway epithelial cells regulate immune reactivity of local dendritic cells (DCs), thus contributing to microenvironment homeostasis. In this study, we set out to identify factors that mediate this regulatory interaction. We show that tracheal epithelial cells secrete soluble factors that downregulate TNF-α and IL-12p40 secretion by bone marrow-derived DCs but upregulate IL-10 and arginase-1. Size exclusion chromatography identified small secreted molecules having high modulatory activity on DCs. We observed that airway tracheal epithelial cells constitutively release the lipid mediator PGE(2). Blocking the synthesis of PGs within airway epithelial cells relieved DCs from inhibition. Cyclooxygenase-2 was found to be expressed in primary tracheal epithelial cell cultures in vitro and in vivo as shown by microdissection of epithelial cells followed by real-time PCR. Paralleling these findings we observed that DCs treated with an antagonist for E-prostanoid 4 receptor as well as DCs lacking E-prostanoid 4 receptor showed reduced inhibition by airway epithelial cells with respect to secretion of proinflammatory cytokines measured by ELISA. Furthermore, PGE(2) mimicked the effects of epithelial cells on DCs. The results indicate that airway epithelial cell-derived PGE(2) contributes to the modulation of DCs under homeostatic conditions.
Assuntos
Brônquios/imunologia , Células Dendríticas/imunologia , Dinoprostona/fisiologia , Regulação para Baixo/imunologia , Mucosa Respiratória/imunologia , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Brônquios/citologia , Brônquios/metabolismo , Proliferação de Células , Células Cultivadas , Células Dendríticas/metabolismo , Dinoprostona/metabolismo , Regulação para Baixo/genética , Feminino , Homeostase/genética , Homeostase/imunologia , Mediadores da Inflamação/antagonistas & inibidores , Mediadores da Inflamação/metabolismo , Mediadores da Inflamação/fisiologia , Subunidade p40 da Interleucina-12/antagonistas & inibidores , Subunidade p40 da Interleucina-12/metabolismo , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucosa Respiratória/citologia , Mucosa Respiratória/metabolismo , Traqueia/imunologia , Traqueia/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/metabolismoRESUMO
IL-12 is a crucial cytokine for dendritic cell-mediated induction of Th 1 cell differentiation. TLR ligands induce IL-12 to differing extents. Stimulation of dendritic cells allowed for the differentiation of three groups of TLRs; potency to induce IL-12 decreased in the order of TLR7/9, TLR3/4, and TLR1/2/6 stimulation. The MAPK, PI3K, and IRF (IFN regulatory factor) signaling pathways could be ruled out to be the cause for the differences in IL-12p40 induction. However, we observed that stimulation of dendritic cells with different TLR ligands resulted in striking differences in the kinetics of NF-kappaB activation. LPS induced a rapid but short-lived activation of RelA, whereas CpG-DNA stimulation resulted in prolonged RelA activity at the IL-12p40 promoter. Only TLR2 and TLR4 ligands were capable of inducing S536 phosphorylation of RelA, which has been proposed to be responsible for early termination of NF-kappaB activation. It is suggested that differences in the kinetics of a common TLR signaling module affect the biological response patterns of various TLRs, with IL-12p40 being a gene that needs prolonged NF-kappaB activation.
Assuntos
Células Dendríticas/imunologia , Regulação da Expressão Gênica/imunologia , Subunidade p40 da Interleucina-12/biossíntese , Transdução de Sinais/imunologia , Receptores Toll-Like/agonistas , Fator de Transcrição RelA/metabolismo , Animais , Western Blotting , Células Dendríticas/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Ensaio de Imunoadsorção Enzimática , Feminino , Imunoprecipitação , Subunidade p40 da Interleucina-12/genética , Ligantes , Camundongos , NF-kappa B/genética , NF-kappa B/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição RelA/genéticaRESUMO
Classical STAT1 activation in response to TLR agonists occurs by phosphorylation of the Y701 and S727 residues through autocrine type I IFN signaling and p38 MAPK signaling, respectively. In this study, we report that the TLR9 agonist CpG DNA induced Ifn-beta mRNA, as well as downstream type I IFN-dependent genes, in a MyD88-dependent manner in mouse myeloid dendritic cells. This pathway was required for maximal TNF and IL-6 secretion, as well as expression of cell surface costimulatory molecules. By contrast, neither A- nor B-type CpG-containing oligonucleotides induced Ifn-beta in mouse bone marrow-derived macrophages (BMM) and a CpG-B oligonucleotide did not induce IFn-beta in the macrophage-like cell line, J774. In BMM, STAT1 was alternatively activated (phosphorylated on S727, but not Y701), and was retained in the cytoplasm in response to CpG DNA. CpG DNA responses were altered in BMM from STAT1(S727A) mice; Il-12p40 and Cox-2 mRNAs were more highly induced, whereas Tlr4 and Tlr9 mRNAs were more repressed. The data suggest a novel inhibitory function for cytoplasmic STAT1 in response to TLR agonists that activate p38 MAPK but do not elicit type I IFN production. Indeed, the TLR7 agonist, R837, failed to induce Ifn-beta mRNA and consequently triggered STAT1 phosphorylation on S727, but not Y701, in human monocyte-derived macrophages. The differential activation of Ifn-beta and STAT1 by CpG DNA in mouse macrophages vs dendritic cells provides a likely mechanism for their divergent roles in priming the adaptive immune response.
Assuntos
Ilhas de CpG/imunologia , Células Dendríticas/imunologia , Regulação para Baixo/imunologia , Interferon beta/biossíntese , Macrófagos/imunologia , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais/imunologia , Receptores Toll-Like/antagonistas & inibidores , Adjuvantes Imunológicos/fisiologia , Animais , Comunicação Autócrina/genética , Comunicação Autócrina/imunologia , Linhagem Celular , Células Dendríticas/metabolismo , Regulação para Baixo/genética , Feminino , Regulação da Expressão Gênica/imunologia , Humanos , Interferon beta/genética , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/deficiência , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/fisiologia , Fosforilação , Fator de Transcrição STAT1/fisiologia , Transdução de Sinais/genética , Receptores Toll-Like/biossíntese , Receptores Toll-Like/fisiologiaRESUMO
Post-translational modifications of histone proteins are major mechanisms that modify chromatin structure and regulate gene expression in eukaryotes. Activation of histone acetyltransferases or inhibition of histone deacetylases (HDACs) is generally believed to allow chromatin to assume a more open state, permitting transcriptional activity. We report here the surprising observation that treatment of murine dendritic cells with the HDAC inhibitors trichostatin A (TSA) or suberoylanilide hydroxamic acid (SAHA) in non-apoptotic concentrations strongly inhibited induction of both interleukin-12 protein p40 (IL-12p40) mRNA and protein upon stimulation of Toll-like receptors (TLRs). Moreover, TLR-mediated up-regulation of costimulatory molecules was also inhibited. Up-regulation of tumour necrosis factor-alpha mRNA and protein in response to TLR agonists was only affected upon prolonged exposure to HDAC inhibitors and regulation of IL-1 beta was not affected. Similar effects were apparent in murine and human macrophages. Regarding the mode of action, HDAC inhibition increased the acetylation status at the IL-12p40 locus. Nevertheless, IL-12p40 chromatin remodelling, binding of Rel-A and IRF1 to the IL-12p40 promoter and transcriptional activation were abrogated. In contrast, HDAC inhibitors had no effects on upstream nuclear factor-kappaB and mitogen-activated protein kinase activation. Thus HDACs positively regulate the expression of a subset of cytokine genes by enabling transcription factor recruitment.
Assuntos
Células Dendríticas/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases , Mediadores da Inflamação/metabolismo , Receptores Toll-Like/imunologia , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Cromatina/metabolismo , Citocinas/biossíntese , Células Dendríticas/imunologia , Regulação da Expressão Gênica/imunologia , Ácidos Hidroxâmicos/farmacologia , Subunidade p40 da Interleucina-12/biossíntese , Subunidade p40 da Interleucina-12/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transcrição Gênica/efeitos dos fármacos , VorinostatRESUMO
Hyperbilirubinemia is a frequent side effect induced by long-term therapy with the antibiotic fusidate. The aim of this study was to elucidate the molecular mechanisms of fusidate-induced hyperbilirubinemia by investigating its influence on hepatic transport systems in the canalicular membrane. Using canalicular membrane vesicles from rat liver, we determined the effect of fusidate on the adenosine 5'-triphosphate (ATP)-dependent transport of substrates of the apical conjugate export pump, multi-drug resistance protein 2 (Mrp2, symbol Abcc2) and the bile salt export pump (Bsep, symbol Abcb11). Fusidate inhibited the ATP-dependent transport of the Mrp2 substrates 17beta-glucuronosyl estradiol and leukotriene C4, and the transport of cholyltaurine by Bsep with Ki values of 2.2+/-0.3, 7.6+/-1.3, and 5.5+/-0.8 microM, respectively. To elucidate the in vivo implication of these findings, the effect of fusidate treatment on the elimination of intravenously administered tracer doses of 17beta-glucuronosyl estradiol and cholyltaurine into bile was studied in rats. Treatment with fusidate (100 micromol/kg body weight) reduced the biliary excretion rate of 17beta-glucuronosyl [3H]estradiol and [3H]cholyltaurine by 75 and 80%, respectively. Extended treatment of rats with fusidate (100 micromol/kg body weight, three times daily i.p. for 3 days) reduced hepatic Mrp2 protein levels by 61% (P<0.001). Our data suggest that there are at least two different mechanisms involved in the impairment of transport processes and hepatobiliary elimination by fusidate, direct inhibition of transport of Mrp2 and Bsep substrates by competitive interaction and impairment by a decreased level of hepatic Mrp2.