RESUMO
Dendritic cells (DC) are required for priming antigen-specific T cells and acquired immunity to many important human pathogens, including Mycobacteriuim tuberculosis (TB) and influenza. However, inappropriate priming of auto-reactive T cells is linked with autoimmune disease. Understanding the molecular mechanisms that regulate the priming and activation of naïve T cells is critical for development of new improved vaccines and understanding the pathogenesis of autoimmune diseases. The serine/threonine kinase IKKα (CHUK) has previously been shown to have anti-inflammatory activity and inhibit innate immunity. Here, we show that IKKα is required in DC for priming antigen-specific T cells and acquired immunity to the human pathogen Listeria monocytogenes. We describe a new role for IKKα in regulation of IRF3 activity and the functional maturation of DC. This presents a unique role for IKKα in dampening inflammation while simultaneously promoting adaptive immunity that could have important implications for the development of new vaccine adjuvants and treatment of autoimmune diseases.
Assuntos
Imunidade Adaptativa/genética , Diferenciação Celular/genética , Células Dendríticas/fisiologia , Quinase I-kappa B/fisiologia , Infecções/imunologia , Transferência Adotiva/métodos , Animais , Diferenciação Celular/imunologia , Células Cultivadas , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/transplante , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Infecções/genética , Inflamação/genética , Inflamação/imunologia , Listeria monocytogenes/imunologia , Listeria monocytogenes/patogenicidade , Listeriose/genética , Listeriose/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
Group B Streptococcus (GBS) is a leading cause of invasive bacterial infections in human newborns and immune-compromised adults. The pore-forming toxin (PFT) ß hemolysin/cytolysin (ßh/c) is a major virulence factor for GBS, which is generally attributed to its cytolytic functions. Here we show ßh/c has immunomodulatory properties on macrophages at sub-lytic concentrations. ßh/c-mediated activation of p38 MAPK drives expression of the anti-inflammatory and immunosuppressive cytokine IL-10, and inhibits both IL-12 and NOS2 expression in GBS-infected macrophages, which are critical factors in host defense. Isogenic mutant bacteria lacking ßh/c fail to activate p38-mediated IL-10 production in macrophages and promote increased IL-12 and NOS2 expression. Furthermore, targeted deletion of p38 in macrophages increases resistance to invasive GBS infection in mice, associated with impaired IL-10 induction and increased IL-12 production in vivo. These data suggest p38 MAPK activation by ßh/c contributes to evasion of host defense through induction of IL-10 expression and inhibition of macrophage activation, a new mechanism of action for a PFT and a novel anti-inflammatory role for p38 in the pathogenesis of invasive bacterial infection. Our studies suggest p38 MAPK may represent a new therapeutic target to blunt virulence and improve clinical outcome of invasive GBS infection.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Hemolisinas/metabolismo , Interleucina-10/biossíntese , Macrófagos/imunologia , Macrófagos/microbiologia , Infecções Estreptocócicas/imunologia , Streptococcus agalactiae/patogenicidade , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Imunidade Inata , Interleucina-12/biossíntese , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Ativação de Macrófagos , Macrófagos/metabolismo , Camundongos , Óxido Nítrico Sintase/biossíntese , Infecções Estreptocócicas/microbiologia , Streptococcus agalactiae/imunologia , Streptococcus agalactiae/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
Inflammation and innate immunity involve signalling pathways leading to the production of inflammatory mediators. Usually such responses are self-limiting, but aberrant resolution of inflammation results in chronic diseases. Much attention has focused on pro-inflammatory signalling but little is known about the mechanisms that resolve inflammation. The IkappaB kinase (IKK) complex contains two catalytic subunits, IKKalpha and IKKbeta, and controls the activation of NF-kappaB transcription factors, which play a pivotal role in inflammation. Ample evidence indicates that IKKbeta mediates NF-kappaB activation in response to pro-inflammatory cytokines and microbial products. IKKalpha regulates an alternative pathway important for lymphoid organogenesis, but the role of IKKalpha in inflammation is unknown. Here we describe a new role for IKKalpha in the negative regulation of macrophage activation and inflammation. IKKalpha contributes to suppression of NF-kappaB activity by accelerating both the turnover of the NF-kappaB subunits RelA and c-Rel, and their removal from pro-inflammatory gene promoters. Inactivation of IKKalpha in mice enhances inflammation and bacterial clearance. Hence, the two IKK catalytic subunits have evolved opposing but complimentary roles needed for the intricate control of inflammation and innate immunity.
Assuntos
Inflamação/metabolismo , Macrófagos/metabolismo , NF-kappa B/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Células Cultivadas , Fibroblastos , Regulação da Expressão Gênica , Quinase I-kappa B , Imunidade Inata/genética , Imunidade Inata/imunologia , Inflamação/genética , Inflamação/microbiologia , Inflamação/patologia , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , NF-kappa B/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-rel/metabolismo , Choque Séptico/induzido quimicamente , Choque Séptico/imunologia , Choque Séptico/microbiologia , Streptococcus agalactiae/imunologia , Streptococcus agalactiae/fisiologia , Análise de Sobrevida , Fator de Transcrição RelARESUMO
Macrophages possess intrinsic tumoricidal activity, yet tumor-associated macrophages (TAMs) rapidly adopt an alternative phenotype within the tumor microenvironment that is marked by tumor-promoting immunosuppressive and trophic functions. The mechanisms that promote such TAM polarization remain poorly understood, but once identified, they may represent important therapeutic targets to block the tumor-promoting functions of TAMs and restore their anti-tumor potential. Here, we have characterized TAMs in a mouse model of metastatic ovarian cancer. We show that ovarian cancer cells promote membrane-cholesterol efflux and depletion of lipid rafts from macrophages. Increased cholesterol efflux promoted IL-4-mediated reprogramming, including inhibition of IFNγ-induced gene expression. Genetic deletion of ABC transporters, which mediate cholesterol efflux, reverts the tumor-promoting functions of TAMs and reduces tumor progression. These studies reveal an unexpected role for membrane-cholesterol efflux in driving TAM-mediated tumor progression while pointing to a potentially novel anti-tumor therapeutic strategy.
Assuntos
Membrana Celular/metabolismo , Reprogramação Celular/fisiologia , Colesterol/metabolismo , Macrófagos/fisiologia , Neoplasias/patologia , Microambiente Tumoral , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Transporte Biológico/fisiologia , Células da Medula Óssea/patologia , Células da Medula Óssea/fisiologia , Células Cultivadas , Progressão da Doença , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias/imunologia , Neoplasias/metabolismo , Evasão Tumoral/fisiologia , Microambiente Tumoral/fisiologiaRESUMO
CD163 is the macrophage receptor for uptake of hemoglobin-haptoglobin complexes. The human receptor can be shed from the macrophage surface owing to a cleavage site for the inflammation-inducible TACE/ADAM17 enzyme. Accordingly, plasma 'soluble CD163' (sCD163) has become a biomarker for macrophage activity and inflammation. The present study disclosed that 10% of sCD163 in healthy persons is actually extracellular vesicle (EV)-associated CD163 not being cleaved and shed. Endotoxin injection of human volunteers caused a selective increase in the ectodomain CD163, while septic patients exhibited high levels of both soluble ectodomain CD163 and extracellular vesicle (EV) CD163, the latter representing up 60% of total plasma CD163. A poor prognosis of septic patients measured as the sequential organ failure assessment (SOFA) score correlated with the increase in membrane-associated CD163. Our results show that soluble ectodomain CD163 and EV CD163 in plasma are part of separate macrophage response in the context of systemic inflammation. While that soluble ectodomain CD163 is released during the acute systemic inflammatory response, this is not the case for EV CD163 that instead may be released during a later phase of the inflammatory response. A separate measurement of the two forms of CD163 constituting 'soluble CD163' in plasma may therefore add to the diagnostic and prognostic value.
Assuntos
Antígenos CD/sangue , Antígenos de Diferenciação Mielomonocítica/sangue , Biomarcadores/sangue , Vesículas Extracelulares/química , Inflamação/sangue , Receptores de Superfície Celular/sangue , Adulto , Idoso , Antígenos CD/química , Antígenos de Diferenciação Mielomonocítica/química , Endotoxinas/administração & dosagem , Endotoxinas/toxicidade , Feminino , Haptoglobinas/química , Haptoglobinas/genética , Voluntários Saudáveis , Hemoglobinas/química , Hemoglobinas/genética , Humanos , Inflamação/induzido quimicamente , Macrófagos/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Prognóstico , Isoformas de Proteínas/genética , Receptores de Superfície Celular/químicaRESUMO
The spleen plays an important role in protective immunity to bloodborne pathogens. Macrophages and dendritic cells (DCs) in the spleen marginal zone capture microbial antigens to trigger adaptive immune responses. Marginal zone macrophages (MZMs) can also act as a replicative niche for intracellular pathogens, providing a platform for mounting the immune response. Here, we describe a role for RANK in the coordinated function of antigen-presenting cells in the spleen marginal zone and triggering anti-viral immunity. Targeted deletion of RANK results in the selective loss of CD169+ MZMs, which provide a niche for viral replication, while RANK signaling in DCs promotes the recruitment and activation of anti-viral memory CD8 T cells. These studies reveal a role for the RANKL/RANK signaling axis in the orchestration of protective immune responses in the spleen marginal zone that has important implications for the host response to viral infection and induction of acquired immunity.
Assuntos
Linfócitos T CD8-Positivos/metabolismo , NF-kappa B/metabolismo , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Animais , Células Dendríticas/metabolismo , Citometria de Fluxo , Imunidade Inata/fisiologia , Macrófagos/metabolismo , Camundongos , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Transdução de Sinais/fisiologia , Baço/metabolismoRESUMO
Cellular stress activates multiple mitogen-activated protein kinase (MAPK) cascades and immediate-early gene (IEG) transcription. To address how these events are linked, we investigated the endogenous signaling/transcription factor network driving IEG activation by arsenite and anisomycin in the human osteosarcoma cell line HOS/TE-85. Induction of IEG transcription by both stresses corresponded temporally with the phosphorylation of the regulatory factors Elk-1 and cAMP response element-binding protein (CREB), along with activation of the extracellular signal-regulated kinase (ERK), stress-activated protein kinase (SAPK) and p38 MAPK cascades. To assess the role of the different cascades, they were selectively inhibited with PD98059, SP600125 and SB203580, respectively. This implicated all three cascades in Elk-1 phosphorylation after arsenite treatment, whereas ERK and SAPK inhibition diminished this, and IEG mRNA levels, downstream of anisomycin. SB blocked phosphorylation of both serum response factor (SRF) and CREB, and strongly reduced IEG activation by both stresses. Combining PD with SB further reduced arsenite induction of IEG transcription. Thus, all three MAPK cascades mediate anisomycin- and arsenite-induced signaling to IEG promoters in HOS cells through the differential targeting of Elk-1, SRF and CREB.
Assuntos
Anisomicina/farmacologia , Arsenitos/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas de Ligação a DNA , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Precoces , Sistema de Sinalização das MAP Quinases , Proteínas Proto-Oncogênicas/metabolismo , Fator de Resposta Sérica/metabolismo , Fatores de Transcrição , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Fosforilação , Ativação Transcricional , Células Tumorais Cultivadas , Proteínas Elk-1 do Domínio etsRESUMO
The nuclear factor kappaB (NF-kappaB) pathway plays a central role in inflammation and immunity. In response to proinflammatory cytokines and pathogen-associated molecular patterns, NF-kappaB activation is controlled by IkappaB kinase (IKK)beta. Using Cre/lox-mediated gene targeting of IKKbeta, we have uncovered a tissue-specific role for IKKbeta during infection with group B streptococcus. Although deletion of IKKbeta in airway epithelial cells had the predicted effect of inhibiting inflammation and reducing innate immunity, deletion of IKKbeta in the myeloid lineage unexpectedly conferred resistance to infection that was associated with increased expression of interleukin (IL)-12, inducible nitric oxide synthase (NOS2), and major histocompatibility complex (MHC) class II by macrophages. We also describe a previously unknown role for IKKbeta in the inhibition of signal transducer and activator of transcription (Stat)1 signaling in macrophages, which is critical for IL-12, NOS2, and MHC class II expression. These studies suggest that IKKbeta inhibits the "classically" activated or M1 macrophage phenotype during infection through negative cross talk with the Stat1 pathway. This may represent a mechanism to prevent the over-exuberant activation of macrophages during infection and contribute to the resolution of inflammation. This establishes a new role for IKKbeta in the regulation of macrophage activation with important implications in chronic inflammatory disease, infection, and cancer.
Assuntos
Quinase I-kappa B/imunologia , Quinase I-kappa B/fisiologia , Inflamação/prevenção & controle , Inflamação/fisiopatologia , Ativação de Macrófagos/fisiologia , Macrófagos/fisiologia , Animais , Deleção de Genes , Antígenos HLA-D/imunologia , Humanos , Quinase I-kappa B/genética , Imunidade Inata , Macrófagos/imunologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Pneumonia Pneumocócica/imunologia , Infecções Estreptocócicas/imunologia , Streptococcus agalactiaeRESUMO
The World Health Organization estimates that lower respiratory tract infections (excluding tuberculosis) account for approximately 35% of all deaths caused by infectious diseases. In many cases, the cause of death may be caused by multiple pathogens, e.g., the life-threatening bacterial pneumonia observed in patients infected with influenza virus. The ability to evolve more efficient immunity on each successive encounter with antigen is the hallmark of the adaptive immune response. However, in the absence of cross-reactive T and B cell epitopes, one lung infection can modify immunity and pathology to the next for extended periods of time. We now report for the first time that this phenomenon is mediated by a sustained desensitization of lung sentinel cells to Toll-like receptor (TLR) ligands; this is an effect that lasts for several months after resolution of influenza or respiratory syncytial virus infection and is associated with reduced chemokine production and NF-kappaB activation in alveolar macrophages. Although such desensitization may be beneficial in alleviating overall immunopathology, the reduced neutrophil recruitment correlates with heightened bacterial load during secondary respiratory infection. Our data therefore suggests that post-viral desensitization to TLR signals may be one possible contributor to the common secondary bacterial pneumonia associated with pandemic and seasonal influenza infection.
Assuntos
Infecções Bacterianas/imunologia , Infecções por Orthomyxoviridae/imunologia , Infecções Respiratórias/imunologia , Receptores Toll-Like/imunologia , Animais , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Citocinas/imunologia , Citocinas/farmacologia , Células Epiteliais/imunologia , Feminino , Flagelina/farmacologia , Ligantes , Lipopolissacarídeos/farmacologia , Macrófagos Alveolares/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , NF-kappa B/imunologia , Neutrófilos/imunologia , Pseudomonas aeruginosa , Sistema Respiratório/imunologia , Streptococcus pneumoniaeRESUMO
Selenium oxyanions, particularly selenite, can be highly toxic to living organisms. Few bacteria reduce both selenate and selenite into the less toxic elemental selenium. Insights into the mechanisms of the transport and the reduction of selenium oxyanions in Escherichia coli were provided by a genetic analysis based on transposon mutagenesis. Ten mutants impaired in selenate reduction were analysed. Three of them were altered in genes encoding transport proteins including a porin, an inner-membrane protein and a sulfate carrier. Two mutants were altered in genes required for molybdopterin biosynthesis, strongly suggesting that the selenate reductase of E. coli is a molybdoenzyme. However, mutants deleted in various oxomolybdenum enzymes described so far in this species still reduced selenate. Finally, a mutant in the gene ygfK encoding a putative oxidoreductase was obtained. This gene is located upstream of ygfN and ygfM in the ygfKLMN putative operon. YgfN and YgfM code for a molybdopterin-containing enzyme and a polypeptide carrying a FAD domain, respectively. It is therefore proposed that the selenate reductase of E. coli is a structural complex including the proteins YgfK, YgfM and YgfN. In addition, all the various mutants were still able to reduce selenite into elemental selenium. This implies that the transport and reduction of this compound are clearly distinct from those of selenate.
Assuntos
Coenzimas , Escherichia coli/enzimologia , Metaloproteínas/metabolismo , Oxirredutases/metabolismo , Pteridinas/metabolismo , Compostos de Selênio/metabolismo , Elementos de DNA Transponíveis , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Metaloproteínas/genética , Cofatores de Molibdênio , Mutagênese Insercional , Óperon , Oxirredução , Oxirredutases/genética , Ácido Selênico , Selenito de Sódio/metabolismoRESUMO
Selenium can provoke contrasting effects on living organisms. It is an essential trace element, and low concentrations have beneficial effects, such as the reduction of the incidence of cancer. However, higher concentrations of selenium salts can be toxic and mutagenic. The bases for both toxicity and protection are not clearly understood. To provide insights into these mechanisms, we analyzed the proteomic response of Escherichia coli cells to selenate and selenite treatment under aerobic conditions. We identified 23 proteins induced by both oxides and ca. 20 proteins specifically induced by each oxide. A striking result was the selenite induction of 8 enzymes with antioxidant properties, particularly the manganese and iron superoxide dismutases (SodA and SodB). The selenium inductions of sodA and sodB were controlled by the transcriptional regulators SoxRS and Fur, respectively. Strains with decreased superoxide dismutase activities were severely impaired in selenium oxide tolerance. Pretreatment with a sublethal selenite concentration triggered an adaptive response dependent upon SoxRS, conferring increased selenite tolerance. Altogether, our data indicate that superoxide dismutase activity is essential for the cellular defense against selenium salts, suggesting that superoxide production is a major mechanism of selenium toxicity under aerobic conditions.
Assuntos
Proteínas de Escherichia coli , Escherichia coli/efeitos dos fármacos , Compostos de Selênio/farmacologia , Superóxido Dismutase/metabolismo , Aerobiose , Proteínas de Bactérias/análise , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Regulação da Expressão Gênica , Mutação , Proteínas Repressoras/genética , Ácido Selênico , Selenito de Sódio/farmacologia , Superóxido Dismutase/análise , Superóxido Dismutase/biossíntese , Transativadores/genética , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
IkappaB Kinase (IKK)alpha is required for activation of an alternative NF-kappaB signaling pathway based on processing of the NF-kappaB2/p100 precursor protein, which associates with RelB in the cytoplasm. This pathway, which activates RelB:p52 dimers, is required for induction of several chemokine genes needed for organization of secondary lymphoid organs. We investigated the basis for the IKKalpha dependence of the induction of these genes in response to engagement of the lymphotoxin beta receptor (LTbetaR). Using chromatin immunoprecipitation, we found that the promoters of organogenic chemokine genes are recognized by RelB:p52 dimers and not by RelA:p50 dimers, the ubiquitous target for the classical NF-kappaB signaling pathway. We identified in the IKKalpha-dependent promoters a novel type of NF-kappaB-binding site that is preferentially recognized by RelB:p52 dimers. This site links induction of organogenic chemokines and other important regulatory molecules to activation of the alternative pathway.