RESUMO
Redox stress related loss of beta cell function is a feature of diabetes. Exposure of beta cells and islets to inflammatory mediators elevates reactive oxygen species (ROS) and beta cell dysfunction. Direct molecular manipulation of NADPH oxidase-1 (NOX-1) has identified a key role for NOX-1 in cytokine-induced beta cell dysfunction. Plasmid driven elevation of NOX-1 resulted in elevated ROS, loss of glucose-stimulated-insulin-secretion and increased apoptosis. These outcomes on beta cell function are analogous to cytokine treatment. In contrast, reduction of NOX-1 expression, by shRNA, conferred protection to beta cells and islets from the damaging effects of inflammatory cytokines. Collectively, these data support the therapeutic potential for NOX-1 inhibition in diabetes.
Assuntos
Apoptose , Citocinas/imunologia , Inflamação/imunologia , Células Secretoras de Insulina/imunologia , Células Secretoras de Insulina/patologia , NADH NADPH Oxirredutases/imunologia , Animais , Linhagem Celular , Diabetes Mellitus/genética , Diabetes Mellitus/imunologia , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Glucose/metabolismo , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Insulina/metabolismo , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Camundongos , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , NADPH Oxidase 1 , Estresse Oxidativo , Interferência de RNA , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio/metabolismo , Regulação para CimaRESUMO
Autologous fat grafting is an effective reconstructive surgery technique; however, its success is limited by inconsistent graft retention and an environment characterized by high oxidative stress and inflammation. Adipose-derived stem cells (ADSCs) increase the survival of fat grafts, although the underlying mechanisms remain unclear. Here, TLR4(-/-) and Nrf2(-/-) mice were used to explore the effects of oxidative stress and inflammation on the viability and function of ADSCs in vitro and in vivo. Enrichment of fat grafts with ADSCs inhibited inflammatory cytokine production, enhanced growth factor levels, increased fat graft survival, downregulated NADPH oxidase (NOX)1 and 4 expression, increased vascularization and reduced ROS production in a manner dependent on toll-like receptor (TLR)-4 and nuclear factor erythroid 2-related factor 2 (Nrf2) expression. Immunohistochemical analysis showed that exposure to hypoxia enhanced ADSC growth and promoted the differentiation of ADSCs into vascular endothelial cells. Hypoxia-induced inflammatory cytokine, growth factor and NOX1/4 upregulation, as well as increased ROS production and apoptosis in ADSCs were dependent on TLR4 and Nrf2, which also modulated the effect of ADSCs on promoting endothelial progenitor cell migration and angiogenesis. Western blot analyses showed that the effects of hypoxia on ADSCs were regulated by crosstalk between Nrf2 antioxidant responses and NF-κB- and TLR4-mediated inflammatory responses. Taken together, our results indicate that ADSCs can increase the survival of fat transplants through the modulation of inflammatory and oxidative responses via Nrf2 and TLR4, suggesting potential strategies to improve the use of ADSCs for cell therapy.
Assuntos
Tecido Adiposo/transplante , Sobrevivência de Enxerto/fisiologia , Células-Tronco Mesenquimais/citologia , Fator 2 Relacionado a NF-E2/genética , Neovascularização Fisiológica/genética , Receptor 4 Toll-Like/genética , Adipócitos/citologia , Adipócitos/imunologia , Tecido Adiposo/citologia , Tecido Adiposo/imunologia , Animais , Diferenciação Celular , Hipóxia Celular , Citocinas/genética , Citocinas/imunologia , Células Endoteliais/citologia , Células Endoteliais/imunologia , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Células-Tronco Mesenquimais/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/imunologia , NADPH Oxidase 1 , NADPH Oxidase 4 , NADPH Oxidases/genética , NADPH Oxidases/imunologia , Fator 2 Relacionado a NF-E2/imunologia , NF-kappa B/genética , NF-kappa B/imunologia , Neovascularização Fisiológica/imunologia , Estresse Oxidativo , Cultura Primária de Células , Transdução de Sinais , Receptor 4 Toll-Like/imunologia , Transplante HomólogoRESUMO
NADPH oxidases (NOXs) are involved in inflammation, angiogenesis, tumor growth, and osteoclast differentiation. However, the role of NOX1 and NOX2 in macrophage differentiation and tumor progression is still elusive. Here we report that NOX1 and NOX2 are critical for the differentiation of monocytes to macrophages, the polarization of M2-type but not M1-type macrophages, and the occurrence of tumor-associated macrophages (TAMs). We found that deletion of both NOX1 and NOX2 led to a dramatic decrease in ROS production in macrophages and resulted in impaired efficiency in monocyte-to-macrophage differentiation and M2-type macrophage polarization. We further showed that NOX1 and NOX2 were critical for the activation of the MAPKs JNK and ERK during macrophage differentiation and that the deficiency of JNK and ERK activation was responsible for the failure of monocyte-to-macrophage differentiation, in turn affecting M2 macrophage polarization. Furthermore, we demonstrated that the decrease in M2 macrophages and TAMs, concomitant with the reduction of cytokine and chemokine secretion, contributed to the delay in wound healing and the inhibition of tumor growth and metastasis in NOX1/2 double knockout mice compared with WT mice. Collectively, these data provide direct evidence that NOX1 and NOX2 deficiency impairs macrophage differentiation and the occurrence of M2-type TAMs during tumor development.
Assuntos
Diferenciação Celular/imunologia , Macrófagos/imunologia , Glicoproteínas de Membrana/imunologia , Monócitos/imunologia , NADH NADPH Oxirredutases/imunologia , NADPH Oxidases/imunologia , Espécies Reativas de Oxigênio/imunologia , Animais , Diferenciação Celular/genética , Quimiocinas/genética , Quimiocinas/imunologia , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/imunologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/imunologia , Macrófagos/enzimologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Monócitos/enzimologia , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , NADPH Oxidase 1 , NADPH Oxidase 2 , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
The liver fluke Fasciola hepatica remains an important agent of food-borne trematode disease producing great economic losses due to its negative effect on productivity of livestock grazing in temperate areas. The prevailing control strategy based on anthelmintic drugs is unsustainable due to widespread resistance hence vaccination appears as an attractive option to pursue. In this study we evaluate the effect of vaccination in calves with a functional recombinant thioredoxin glutathione reductase (rFhTGR) from liver fluke, a critical antioxidant enzyme at the crossroads of the thioredoxin and glutathione metabolism in flatworms. The recombinant enzyme produced in Escherichia coli was tested in two vaccination experiments; in the first trial rFhTGR was administered in combination with FreundÌs Incomplete Adjuvant (FIA) in a three-inoculation scheme on weeks 0, 4 and 8; in the second trial rFhTGR was given mixed with Adyuvac 50 or Alum as adjuvants on weeks 0 and 4. In both cases calves were challenged with metacercariae (400 in the first and 500 in the second trial) 2 weeks after the last inoculation. Our results demonstrate that two or three doses of the vaccine induced a non-significant reduction in worm counts of 8.2% (FIA), 3.8% (Adyuvac 50) and 23.0% (Alum) compared to adjuvant controls indicating that rFhTGR failed to induce a protective immunity in challenged calves. All vaccine formulations induced a mixed IgG1/IgG2 response but no booster was observed after challenge. No correlations between antibody titres and worm burdens were found.
Assuntos
Antígenos de Helmintos/imunologia , Doenças dos Bovinos/prevenção & controle , Fasciolíase/veterinária , Imunização/veterinária , Complexos Multienzimáticos/imunologia , NADH NADPH Oxirredutases/imunologia , Adjuvantes Imunológicos , Animais , Bovinos , Fasciola hepatica/enzimologia , Fasciola hepatica/imunologia , Fasciolíase/prevenção & controle , Imunização/normas , Carga Parasitária , Proteínas Recombinantes/imunologiaRESUMO
The role of the reactive oxygen species-producing NADPH oxidase family of enzymes in the pathology of influenza A virus infection remains enigmatic. Previous reports implicated NADPH oxidase 2 in influenza A virus-induced inflammation. In contrast, NADPH oxidase 1 (Nox1) was reported to decrease inflammation in mice within 7 days post-influenza A virus infection. However, the effect of NADPH oxidase 1 on lethality and adaptive immunity after influenza A virus challenge has not been explored. Here we report improved survival and decreased morbidity in mice with catalytically inactive NADPH oxidase 1 (Nox1*/Y) compared with controls after challenge with A/PR/8/34 influenza A virus. While changes in lung inflammation were not obvious between Nox1*/Y and control mice, we observed alterations in the T cell response to influenza A virus by day 15 post-infection, including increased interleukin-7 receptor-expressing virus-specific CD8+ T cells in lungs and draining lymph nodes of Nox1*/Y, and increased cytokine-producing T cells in lungs and spleen. Furthermore, a greater percentage of conventional and interstitial dendritic cells from Nox1*/Y draining lymph nodes expressed the co-stimulatory ligand CD40 within 6 days post-infection. Results indicate that NADPH oxidase 1 modulates the innate and adaptive cellular immune response to influenza virus infection, while also playing a role in host survival. Results suggest that NADPH oxidase 1 inhibitors may be beneficial as adjunct therapeutics during acute influenza infection.
Assuntos
Imunidade Adaptativa , Linfócitos T CD8-Positivos/imunologia , Imunidade Inata , Vírus da Influenza A/imunologia , NADH NADPH Oxirredutases/imunologia , Infecções por Orthomyxoviridae/imunologia , Animais , Ligante de CD40/genética , Ligante de CD40/imunologia , Células Dendríticas/imunologia , Masculino , Camundongos , Camundongos Transgênicos , NADH NADPH Oxirredutases/genética , NADPH Oxidase 1 , Infecções por Orthomyxoviridae/genéticaRESUMO
Interferon gamma (IFNγ)-producing CD8(+) T cells (Tc1) play important roles in immunological disease. We now report that CD3/CD28-mediated stimulation of CD8(+) T cells to generate Tc1 cells, not only increases IFNγ production but also boosts the generation of reactive oxygen species (ROS) and augments expression of CD39. Inhibition of NADPH oxidases or knockdown of gp91phox in CD8(+) T cells abrogates ROS generation, which in turn modulates JNK and NFκB signalling with decreases in both IFNγ levels and CD39 expression. CD39(+)CD8(+) T cells substantially inhibit IFNγ production by CD39(-)CD8(+) T cells via the paracrine generation of adenosine, which is operational via adenosine type 2A receptors. Increases in numbers of CD39(+)CD8(+) T cells and associated enhancements in ROS signal transduction are noted in cells from patients with Crohn's disease. Our findings provide insights into Tc1-mediated IFNγ responses and ROS generation and link these pathways to CD39/adenosine-mediated effects in immunological disease.
Assuntos
Adenosina/metabolismo , Antígenos CD/imunologia , Apirase/imunologia , Colo/imunologia , Doença de Crohn/imunologia , Interferon gama/imunologia , Leucócitos Mononucleares/imunologia , Complexos Multienzimáticos/imunologia , NADH NADPH Oxirredutases/imunologia , NADPH Oxidases/imunologia , Linfócitos T Citotóxicos/imunologia , Adulto , Idoso , Antígenos CD/metabolismo , Apirase/metabolismo , Western Blotting , Antígenos CD28 , Complexo CD3 , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Imunoprecipitação da Cromatina , Colo/citologia , Feminino , Citometria de Fluxo , Humanos , Imunossupressores , Interferon gama/metabolismo , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Masculino , Pessoa de Meia-Idade , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/metabolismo , NADPH Oxidases/metabolismo , NF-kappa B/imunologia , NF-kappa B/metabolismo , Espécies Reativas de Oxigênio , Receptores Purinérgicos P1/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T Citotóxicos/metabolismo , Adulto JovemRESUMO
Peptides presented by MHC class I molecules are mostly derived from proteins synthesized by the antigen-presenting cell itself, while peptides presented by MHC class II molecules are predominantly from materials acquired by endocytosis. External antigens can also be presented by MHC class I molecules in a process referred to as cross-presentation. Here, we report that mouse dendritic cell (DC) engagement to a phagocytic target alters endocytic processing and inhibits the proteolytic activities. During phagocytosis, endosome maturation is delayed, shows less progression toward the lysosome, and the endocytosed soluble antigen is targeted for MHC class I cross-presentation. The antigen processing in these arrested endosomes is under the control of NAPDH oxidase associated ROS. We also show that cathepsin S is responsible for the generation of the MHC class I epitope. Taken together, our results suggest that in addition to solid structure uptake, DC phagocytosis simultaneously modifies the kinetics of endosomal trafficking and maturation. As a consequence, external soluble antigens are targeted into the MHC class I cross-presentation pathway.
Assuntos
Apresentação de Antígeno , Apresentação Cruzada , Células Dendríticas/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Fagocitose , Animais , Catepsinas/imunologia , Catepsinas/metabolismo , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Endocitose , Endossomos/imunologia , Endossomos/metabolismo , Epitopos/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Inflamação/induzido quimicamente , Inflamação/imunologia , Inflamação/patologia , Lisossomos/imunologia , Lisossomos/metabolismo , Camundongos , Camundongos Knockout , Complexos Multienzimáticos/imunologia , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/imunologia , NADH NADPH Oxirredutases/metabolismo , Ovalbumina/imunologia , Ovalbumina/farmacologia , Cultura Primária de Células , Espécies Reativas de Oxigênio/metabolismoRESUMO
PURPOSE OF REVIEW: The aim of the present review was to discuss the effects of pollen components on innate immune responses. RECENT FINDINGS: Pollens contain numerous factors that can stimulate an innate immune response. These include intrinsic factors in pollens such as nicotinamide adenine dinucleotide phosphate oxidases, proteases, aqueous pollen proteins, lipids, and antigens. Each component stimulates innate immune response in a different manner. Pollen nicotinamide adenine dinucleotide phosphate oxidases induce reactive oxygen species generation and recruit neutrophils that stimulate subsequent allergic inflammation. Pollen proteases damage epithelial barrier function and increase antigen uptake. Aqueous pollen extract proteins and pollen lipids modulate dendritic cell function and induce Th2 polarization. Clinical studies have shown that modulation of innate immune response to pollens with toll-like receptor 9- and toll-like receptor 4-stimulating conjugates is well tolerated and induces clear immunological effects, but is not very effective in suppressing primary clinical endpoints of allergic inflammation. SUMMARY: Additional research on innate immune pathways induced by pollen components is required to develop novel strategies that will mitigate the development of allergic inflammation.
Assuntos
Imunidade Inata , NADH NADPH Oxirredutases/metabolismo , Neutrófilos/imunologia , Pólen/metabolismo , Rinite Alérgica Sazonal/imunologia , Células Th2/imunologia , Animais , Antígenos de Plantas/imunologia , Humanos , NADH NADPH Oxirredutases/imunologia , Pólen/imunologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
OBJECTIVE: To study the immunogenicity and the immuno-protection of thioredoxin glutathione reductase from Schistosomajaponicum (SjTGR) against schistosome infection in mice. METHODS: Seventy-five mice were randomly divided into 5 groups, namely, blank group, PBS group, CpG2 immunized group, TGR immunized group and TGR + CpG2 co-immunized group. Each mouse was immunized for 3 times. The mice were tail bled before the first immunization and 2 weeks after the third immunization. The serum antibody levels of total IgG, IgG1 and IgG2a against SjTGR were assayed by ELISA. Two weeks after the third immunization, each mouse was infected with 40 ± 2 S. japonicum cercariae by abdominal skin penetration. Forty-two days later, all the mice were sacrificed to collect schistosome adult worms and liver eggs. The worm and egg reduction rates were calculated respectively. The single splenocyte of mouse was collected 2 weeks after the third immunization, and the expressions of CD44high, CD4+CD44high or CD8+CD44high on splenocytes of mice were examined by flow cytometry. After 72 h incubation with recombinant SjTGR, the levels of IL-2, IL-4, IL-10, and IFN-γ in the single-cell supernatant were determined by using ELISA kit. RESULTS: Two weeks after the third immunization, the titers of serum IgG against SjTGR in mice immunized with SjTGR and co-immunized with SjTGR and CpG2 were higher than 1:200 000. The IgG2a: IgG1 ratio (IgG2a/IgG1) increased slowly with time in both TGR immunized group and TGR + CpG2 co-immunized group. There were obviously higher levels of IFN-γ and IL-2 in the cell supernatant in the TGR immunized group and TGR + CpG2 co-immunized group compared to the blank, PBS and CpG2 groups (P < 0.05). The increased subpopulations of CD44high, CD8+CD44high and CD4+ CD44high cells in the splenocytes from mice immunized by SjTGR and co-immunized by SjTGR and CpG2 were found comparing to the blank, PBS and CpG2 groups (P < 0.05). The TGR immunization and TGR + CpG2 co- immunization caused 9.4% and 10.5% reductions in the number of adult worms and 9.2% and 32.8% reductions in the number of eggs, respectively. CONCLUSIONS: SjTGR displays strong immunogenicity inducing Th1 type immune response in mice. However, it could not produce protective efficacy against S. japonicum infection. CpG2 ODN may be a broadly effective Th1 adjuvant.
Assuntos
Complexos Multienzimáticos/imunologia , NADH NADPH Oxirredutases/imunologia , Schistosoma japonicum/imunologia , Animais , Anticorpos Anti-Helmínticos/sangue , Citocinas/análise , Feminino , Imunização , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos BALB C , Oligodesoxirribonucleotídeos/farmacologia , Schistosoma japonicum/enzimologia , Células Th1/imunologiaRESUMO
Oxygen-derived free radicals, collectively termed reactive oxygen species (ROS), play important roles in immunity, cell growth, and cell signaling. In excess, however, ROS are lethal to cells, and the overproduction of these molecules leads to a myriad of devastating diseases. The key producers of ROS in many cells are the NOX family of NADPH oxidases, of which there are seven members, with various tissue distributions and activation mechanisms. NADPH oxidase is a multisubunit enzyme comprising membrane and cytosolic components, which actively communicate during the host responses to a wide variety of stimuli, including viral and bacterial infections. This enzymatic complex has been implicated in many functions ranging from host defense to cellular signaling and the regulation of gene expression. NOX deficiency might lead to immunosuppression, while the intracellular accumulation of ROS results in the inhibition of viral propagation and apoptosis. However, excess ROS production causes cellular stress, leading to various lethal diseases, including autoimmune diseases and cancer. During the later stages of injury, NOX promotes tissue repair through the induction of angiogenesis and cell proliferation. Therefore, a complete understanding of the function of NOX is important to direct the role of this enzyme towards host defense and tissue repair or increase resistance to stress in a timely and disease-specific manner.
Assuntos
Doenças do Sistema Imunitário/imunologia , Infecções/imunologia , NADH NADPH Oxirredutases/metabolismo , Animais , Dano ao DNA , Humanos , Imunidade Inata , Conformação Molecular , NADH NADPH Oxirredutases/imunologia , Neovascularização Fisiológica , Espécies Reativas de Oxigênio/metabolismo , Relação Estrutura-Atividade , CicatrizaçãoRESUMO
In visceral leishmaniasis (VL), Th1-type of immune responses play an important role which correlates with recovery from and resistance to disease resulting in lifelong immunity. Based on this rationale, the soluble leishmanial antigens that elicit cellular responses in peripheral blood mononuclear cells (PBMCs) from cured Leishmania patients were characterized through immunoproteomic approach which led to the identification of trypanothione reductase (TPR) (a cytosolic enzyme explored as a drug target), as one of the potent Th1 stimulatory protein. In this study, the immunogenicity of recombinant Leishmania donovani TPR (rLdTPR) was assessed in PBMCs of cured Leishmania-infected patients/hamsters and further evaluated its prophylactic efficacy against L. donovani challenges in hamsters. Substantial proliferative responses to rLdTPR, as compared to soluble L. donovani antigen, were observed in Leishmania-infected cured patients as well as in hamsters. Moreover, rLdTPR reasonably stimulated PBMCs of cured Leishmania patients to produce IFNγ, IL-12, and TNF-α but not IL-4 or IL-10. On the other hand, the protein downregulated LPS-induced IL-10 as well as soluble L. donovani antigen-induced IL-4 production in PBMCs of Leishmania patients. In case of cured hamsters, rLdTPR generates mixed Th1 and Th2 immune response. Vaccination with rLdTPR along with Bacillus Calmette-Guerin (BCG) was able to provide considerably good prophylactic efficacy (~60%) against L. donovani challenge in hamsters. The efficacy was supported by the increased inducible NO synthase mRNA transcript and Th1-type cytokines IFNγ, IL-12, and TNF-α and downregulation of IL-4, IL-10, and TGF-ß. Since rLdTPR protein is an important target, further attempts towards determination of immunodominant regions for designing fusion peptides may be taken up to optimize its prophylactic efficacy.
Assuntos
Antígenos de Protozoários/imunologia , Leishmania donovani/enzimologia , Leishmaniose Visceral/prevenção & controle , NADH NADPH Oxirredutases/imunologia , Vacinas Protozoárias/imunologia , Células Th1/imunologia , Adolescente , Adulto , Animais , Formação de Anticorpos , Criança , Pré-Escolar , Cricetinae , Citocinas/imunologia , Feminino , Humanos , Lactente , Leishmania donovani/genética , Leishmania donovani/imunologia , Leishmaniose Visceral/imunologia , Leucócitos Mononucleares/imunologia , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes/imunologia , Vacinação , Adulto JovemRESUMO
Proanthocyanidins (PAs) contribute to poplar defense mechanisms against biotic and abiotic stresses. Transcripts of PA biosynthetic genes accumulated rapidly in response to infection by the fungus Marssonina brunnea f.sp. multigermtubi, treatments of salicylic acid (SA) and wounding, resulting in PA accumulation in poplar leaves. Anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) are two key enzymes of the PA biosynthesis that produce the main subunits: (+)-catechin and (-)-epicatechin required for formation of PA polymers. In Populus, ANR and LAR are encoded by at least two and three highly related genes, respectively. In this study, we isolated and functionally characterized genes PtrANR1 and PtrLAR1 from P. trichocarpa. Phylogenetic analysis shows that Populus ANR1 and LAR1 occurr in two distinct phylogenetic lineages, but both genes have little difference in their tissue distribution, preferentially expressed in roots. Overexpression of PtrANR1 in poplar resulted in a significant increase in PA levels but no impact on catechin levels. Antisense down-regulation of PtrANR1 showed reduced PA accumulation in transgenic lines, but increased levels of anthocyanin content. Ectopic expression of PtrLAR1 in poplar positively regulated the biosynthesis of PAs, whereas the accumulation of anthocyanin and flavonol was significantly reduced (P<0.05) in all transgenic plants compared to the control plants. These results suggest that both PtrANR1 and PtrLAR1 contribute to PA biosynthesis in Populus.
Assuntos
Regulação da Expressão Gênica de Plantas , NADH NADPH Oxirredutases/genética , Folhas de Planta/enzimologia , Proteínas de Plantas/genética , Raízes de Plantas/enzimologia , Populus/enzimologia , Antocianinas/metabolismo , Catequina/metabolismo , DNA Complementar/genética , DNA Complementar/metabolismo , NADH NADPH Oxirredutases/imunologia , NADH NADPH Oxirredutases/metabolismo , Filogenia , Folhas de Planta/genética , Folhas de Planta/imunologia , Proteínas de Plantas/imunologia , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/imunologia , Populus/genética , Populus/imunologia , Proantocianidinas/biossíntese , Proantocianidinas/imunologiaRESUMO
The initial event in disease caused by S. pneumoniae is adhesion of the bacterium to respiratory epithelial cells, mediated by surface expressed molecules including cell-wall proteins. NADH oxidase (NOX), which reduces free oxygen to water in the cytoplasm, was identified in a non-lectin enriched pneumococcal cell-wall fraction. Recombinant NOX (rNOX) was screened with sera obtained longitudinally from children and demonstrated age-dependent immunogenicity. NOX ablation in S. pneumoniae significantly reduced bacterial adhesion to A549 epithelial cells in vitro and their virulence in the intranasal or intraperitoneal challenge models in mice, compared to the parental strain. Supplementation of Δnox WU2 with the nox gene restored its virulence. Saturation of A549 target cells with rNOX or neutralization of cell-wall residing NOX using anti-rNOX antiserum decreased adhesion to A549 cells. rNOX-binding phages inhibited bacterial adhesion. Moreover, peptides derived from the human proteins contactin 4, chondroitin 4 sulfotraferase and laminin5, homologous to the insert peptides in the neutralizing phages, inhibited bacterial adhesion to the A549 cells. Furthermore, rNOX immunization of mice elicited a protective immune response to intranasal or intraperitoneal S. pneumoniae challenge, whereas pneumococcal virulence was neutralized by anti-rNOX antiserum prior to intraperitoneal challenge. Our results suggest that in addition to its enzymatic activity, NOX contributes to S. pneumoniae virulence as a putative adhesin and thus peptides derived from its target molecules may be considered for the treatment of pneumococcal infections. Finally, rNOX elicited a protective immune response in both aerobic and anaerobic environments, which renders NOX a candidate for future pneumococcal vaccine.
Assuntos
Adesinas Bacterianas/metabolismo , Imunidade , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/metabolismo , Streptococcus pneumoniae/enzimologia , Adesinas Bacterianas/genética , Adesinas Bacterianas/imunologia , Aerobiose , Envelhecimento/imunologia , Anaerobiose , Animais , Aderência Bacteriana/efeitos dos fármacos , Vacinas Bacterianas/genética , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/metabolismo , Linhagem Celular , Pré-Escolar , Feminino , Deleção de Genes , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Humanos , Lactente , Camundongos , Complexos Multienzimáticos/deficiência , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/imunologia , NADH NADPH Oxirredutases/deficiência , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/imunologia , Peptídeos/farmacologia , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/imunologiaRESUMO
Mast cells are effector cells that mediate the allergic response through Ag stimulation of IgE bound to FcεRI. In allergic reactions, cross-linking of the surface receptors for IgE on mast cells results in the synthesis of Th2 cytokines such as IL-4 and IL-13, which are critical for the initiation and progression of the allergic response. Despite the important roles of these cytokines, the signaling mechanism by which Ag stimulation mediates the production of IL-4 and IL-13 in mast cells is not clearly understood. In the present study, we found that Ag-stimulated bone marrow-derived mast cells (BMMCs) highly upregulated the expression of BLT2, a leukotriene B(4) receptor, and that blockade of BLT2 with the specific antagonist LY255283 or small interfering RNA knockdown completely abolished the production of Th2 cytokines. Furthermore, BMMCs overexpressing BLT2 showed significantly enhanced production of Th2 cytokines compared with wild-type BMMCs. Additionally, we found that the generation of Nox1-derived reactive oxygen species occurs downstream of BLT2, thus mediating the synthesis of Th2 cytokines. Taken together, our results suggest that the BLT2-Nox1-reactive oxygen species cascade is a previously unsuspected mediatory signaling mechanism to Th2 cytokine production in Ag-stimulated BMMCs, thus contributing to allergic response.
Assuntos
Citocinas/biossíntese , Regulação da Expressão Gênica/imunologia , Mastócitos/metabolismo , Receptores do Leucotrieno B4/metabolismo , Transdução de Sinais/imunologia , Alérgenos/imunologia , Animais , Apresentação de Antígeno , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Separação Celular , Citocinas/imunologia , Feminino , Citometria de Fluxo , Expressão Gênica , Hipersensibilidade/imunologia , Hipersensibilidade/metabolismo , Interleucina-3/biossíntese , Interleucina-3/imunologia , Interleucina-4/biossíntese , Interleucina-4/imunologia , Mastócitos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , NADH NADPH Oxirredutases/imunologia , NADH NADPH Oxirredutases/metabolismo , NADPH Oxidase 1 , Interferência de RNA , Espécies Reativas de Oxigênio , Receptores do Leucotrieno B4/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para CimaRESUMO
One of the hallmarks of Human Immunodeficiency Virus-1 (HIV-1) infection is progressive depletion of the infected and bystander CD4+ T-cells by apoptosis. Different mitochondrial proteins have been implicated in this apoptotic process; however, the role of different subunits of mitochondrial oxidative phosphorylation (OXPHOS) complexes in apoptosis is not clearly understood. Some of the OXPHOS complex subunits seem to perform other functions in addition to their primary role in energy generating process. GRIM-19 (gene associated with retinoid-interferon-induced-mortality-19), a subunit of mitochondrial complex-I was previously implicated in Interferon-ß and retionoic acid induced apoptosis in many tumor cells. In this study we report, using differential gene expression analysis, that GRIM-19 is up-regulated in HIV-1 infected apoptotic T-cells. A temporal up regulation of this subunit was observed in different HIV-1 infected T-cell lines and human PBMC and the extent of increase correlated to increasing apoptosis and virus production. Moreover, silencing GRIM-19 in HIV-1 infected cells reduced apoptosis, indicating its involvement in HIV-1 induced T-cell death.
Assuntos
Proteínas Reguladoras de Apoptose , Apoptose , Complexo I de Transporte de Elétrons/metabolismo , Regulação Enzimológica da Expressão Gênica/genética , Infecções por HIV/enzimologia , Infecções por HIV/imunologia , Mitocôndrias/enzimologia , Mitocôndrias/virologia , NADH NADPH Oxirredutases , Linfócitos T/enzimologia , Linfócitos T/virologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/imunologia , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Clonagem Molecular , Complexo I de Transporte de Elétrons/genética , Regulação Enzimológica da Expressão Gênica/imunologia , Inativação Gênica , Infecções por HIV/fisiopatologia , Infecções por HIV/virologia , HIV-1/imunologia , HIV-1/fisiologia , Humanos , Mitocôndrias/genética , Mitocôndrias/imunologia , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/imunologia , NADH NADPH Oxirredutases/metabolismo , Fosforilação Oxidativa , Estaurosporina/farmacologia , Linfócitos T/citologia , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/farmacologia , Regulação para CimaRESUMO
Foam cell formation is the most important process in atherosclerosis, and low density lipoprotein oxidation by reactive oxygen species (ROS) is the key step in the conversion of macrophages to foam cells. This study reveals the control mechanism of the gene for NADPH oxidase 1 (Nox1), which produces ROS in the formation of foam cells by stimulating TLR4. Treatment of macrophages by the TLR4 agonist LPS stimulated ROS production and ROS-mediated macrophage to foam cell conversion. This LPS-induced ROS production and foam cell formation could be abrogated by pretreatment of macrophages with N-acetyl cysteine or apocynin. LPS increased Nox1 promoter activity, and resultant expression of mRNA and protein. Small interfering RNA mediated inhibition of Nox1 expression decreased LPS-induced ROS production and foam cell formation. LPS-mediated Nox1 expression and the responses occurred in a calcium-independent phospholipase A(2) (iPLA(2))-dependent manner. The iPLA(2)beta-specific inhibitor S-BEL or iPLA(2)beta small interfering RNA attenuated LPS-induced Nox1 expression, ROS production, and foam cell formation. In addition, activation of iPLA(2)beta by LPS caused Akt phosphorylation and was followed by increased Nox1 expression. These results suggest that the binding of LPS and TLR4 increases Nox1 expression through the iPLA(2)beta-Akt signaling pathway, and control ROS production and foam cell formation.
Assuntos
Células Espumosas/imunologia , Regulação da Expressão Gênica/imunologia , Fosfolipases A2 do Grupo IV/imunologia , NADH NADPH Oxirredutases/imunologia , Proteínas Proto-Oncogênicas c-akt/imunologia , Transdução de Sinais/imunologia , Animais , Aterosclerose/imunologia , Aterosclerose/metabolismo , Western Blotting , Sinalização do Cálcio/imunologia , Linhagem Celular , Citometria de Fluxo , Células Espumosas/metabolismo , Expressão Gênica , Fosfolipases A2 do Grupo IV/metabolismo , Lipopolissacarídeos/imunologia , Camundongos , Microscopia Confocal , NADH NADPH Oxirredutases/metabolismo , NADPH Oxidase 1 , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/metabolismoRESUMO
We here present the first genetic fine mapping of experimental autoimmune neuritis (EAN), the animal model of Guillain-Barré syndrome, in a rat advanced intercross line. We identified and refined a total of five quantitative trait loci on rat chromosomes 4, 10, and 12 (RNO4, RNO10, RNO12), showing linkage to splenic IFN-gamma secretion and disease severity. All quantitative trait loci were shared with other models of complex inflammatory diseases. The quantitative trait locus showing strongest linkage to clinical disease was Ean6 and spans 4.3 Mb on RNO12, harboring the neutrophil cytosolic factor 1 (Ncf1) among other genes. Polymorphisms in Ncf1, a member of the NADPH oxidase complex, have been associated with disease regulation in experimental arthritis and encephalomyelitis. We therefore tested the Ncf1 pathway by treating rats with a NADPH oxidase complex activator and ameliorated EAN compared the oil-treated control group. By proving the therapeutic effect of stimulating the NADPH oxidase complex, our data strongly suggest the first identification of a gene regulating peripheral nervous system inflammation. Taken together with previous reports, our findings suggest a general role of Ncf1 and oxidative burst in pathogenesis of experimental autoimmune animal models.
Assuntos
Mapeamento Cromossômico , NADPH Oxidases/genética , Neurite Autoimune Experimental/genética , Neurite Autoimune Experimental/patologia , Animais , Autoanticorpos/sangue , Autoanticorpos/imunologia , Autoantígenos/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Ligação Genética , Genótipo , Síndrome de Guillain-Barré/genética , Síndrome de Guillain-Barré/imunologia , Síndrome de Guillain-Barré/patologia , Interferon gama/imunologia , Interferon gama/metabolismo , Masculino , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/imunologia , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/imunologia , NADH NADPH Oxirredutases/metabolismo , Neurite Autoimune Experimental/imunologia , Fitol/farmacologia , Polimorfismo Genético , Locos de Características Quantitativas , Ratos , Explosão Respiratória/fisiologiaRESUMO
Lymphocytes migrate from the blood into tissue by binding to and migrating across endothelial cells. One of the endothelial cell adhesion molecules that mediate lymphocyte binding is VCAM-1. We have reported that binding to VCAM-1 activates endothelial cell NADPH oxidase for the generation of reactive oxygen species (ROS). The ROS oxidize and stimulate an increase in protein kinase C (PKC)alpha activity. Furthermore, these signals are required for VCAM-1-dependent lymphocyte migration. In this report, we identify a role for protein tyrosine phosphatase 1B (PTP1B) in the VCAM-1 signaling pathway. In primary cultures of endothelial cells and endothelial cell lines, Ab cross-linking of VCAM-1 stimulated an increase in serine phosphorylation of PTP1B, the active form of PTP1B. Ab cross-linking of VCAM-1 also increased activity of PTP1B. This activation of PTP1B was downstream of NADPH oxidase and PKCalpha in the VCAM-1 signaling pathway as determined with pharmacological inhibitors and antisense approaches. In addition, during VCAM-1 signaling, ROS did not oxidize endothelial cell PTP1B. Instead PTP1B was activated by serine phosphorylation. Importantly, inhibition of PTP1B activity blocked VCAM-1-dependent lymphocyte migration across endothelial cells. In summary, VCAM-1 activates endothelial cell NADPH oxidase to generate ROS, resulting in oxidative activation of PKCalpha and then serine phosphorylation of PTP1B. This PTP1B activity is necessary for VCAM-1-dependent transendothelial lymphocyte migration. These data show, for the first time, a function for PTP1B in VCAM-1-dependent lymphocyte migration.
Assuntos
Movimento Celular/fisiologia , Células Endoteliais/enzimologia , Linfócitos/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Transdução de Sinais/fisiologia , Molécula 1 de Adesão de Célula Vascular/metabolismo , Animais , Adesão Celular/fisiologia , Linhagem Celular , Células Endoteliais/citologia , Células Endoteliais/imunologia , Linfócitos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Complexos Multienzimáticos/imunologia , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/imunologia , NADH NADPH Oxirredutases/metabolismo , Oxirredução , Proteína Quinase C-alfa/imunologia , Proteína Quinase C-alfa/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 1 , Proteínas Tirosina Fosfatases/imunologia , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , Molécula 1 de Adesão de Célula Vascular/imunologiaRESUMO
The importance of reactive oxygen intermediate (ROI) production in antimicrobial responses is demonstrated in human patients who suffer from chronic granulomatous disease (CGD) due to defective NADPH oxidase function. Exactly how bacterial products activating Toll-like receptors (TLRs) induce oxidative burst is unknown. Here, we identify the Vav family of Rho guanine nucleotide exchange factors (GEFs) as critical mediators of LPS-induced MyD88-dependent activation of Rac2, NADPH oxidase, and ROI production using mice deficient in Vav1, Vav2, and Vav3. Vav proteins are also required for p38 MAPK activation and for normal regulation of proinflammatory cytokine production, but not for other MyD88-controlled effector pathways such as those involving JNK, COX2, or iNOS and the production of reactive nitrogen intermediates (RNIs). Thus, our data indicate that Vav specifically transduces a subset of signals emanating from MyD88.
Assuntos
Macrófagos/imunologia , Fator 88 de Diferenciação Mieloide/imunologia , Proteínas Proto-Oncogênicas c-vav/imunologia , Espécies Reativas de Oxigênio/imunologia , Explosão Respiratória/imunologia , Transdução de Sinais/imunologia , Animais , Células Cultivadas , Ciclo-Oxigenase 2/imunologia , Citocinas/imunologia , Doença Granulomatosa Crônica/enzimologia , Doença Granulomatosa Crônica/imunologia , Humanos , Inflamação/imunologia , Lipopolissacarídeos/farmacologia , MAP Quinase Quinase 4/imunologia , Macrófagos/enzimologia , Camundongos , Camundongos Knockout , Complexos Multienzimáticos/deficiência , Complexos Multienzimáticos/imunologia , Fator 88 de Diferenciação Mieloide/deficiência , NADH NADPH Oxirredutases/deficiência , NADH NADPH Oxirredutases/imunologia , Óxido Nítrico Sintase Tipo II/imunologia , Proteínas Proto-Oncogênicas c-vav/deficiência , Explosão Respiratória/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteínas rac de Ligação ao GTP/imunologia , Proteína RAC2 de Ligação ao GTPRESUMO
In C(4) plants, granal mesophyll (MS) chloroplasts contain higher photosystem (PS) II and lower PS I activity than agranal bundle sheath (BS) chloroplasts. The maize NAD(P)H dehydrogenase or NAD(P)H-plastoquinone oxidoreductase (also named Ndh complex) from MS and BS chloroplasts, contains at least 11 subunits (NdhA-K) and is homologous to NADH dehydrogenase or Complex I from mitochondria and bacteria. The amount of Ndh complex is higher in BS compared with MS chloroplasts. However, there is little information about the interdependence of the PS II and Ndh complex in chlororespiration and linear and cyclic electron transport in C(4) plants. To characterize the expression of the PS II and Ndh complex in maize plastids, we used cytochrome b559 (cyt b559) antibodies and Ndh immunoglobulins (IgG) to analyze the Ndh complex and PS II in both MS and BS chloroplasts from maize leaves by Western blotting and immunolabeling. In Western blot experiments, it was found that the amount of cyt b559 (a marker for PS II) is 7-8 times higher in MS than BS chloroplasts. Conversely, the NdhH, -J, -K and -E content is 2.5-3 times higher in BS than MS chloroplasts. Similar results were obtained in immunolabeling experiments using Ndh IgGs and cyt b559 antibodies in MS and BS chloroplasts. These data suggest that in BS chloroplasts, ATP could be produced mainly by cyclic electron transport around PS I and Ndh complexes. Conversely, the linear electron transport in BS chloroplasts via PS II could have a lower production of ATP. These results also suggest that the contribution of the Ndh complex in the production of ATP in MS chloroplasts is minimal and that instead, this complex could have a chlororespiratory role.