RESUMO
Sepsis is one of the most challenging health problems worldwide. Here we found that phagocytes from patients with sepsis had considerable upregulation of Toll-like receptor 4 (TLR4) and TLR2; however, shock-inducing inflammatory responses mediated by these TLRs were inhibited by ES-62, an immunomodulator secreted by the filarial nematode Acanthocheilonema viteae. ES-62 subverted TLR4 signaling to block TLR2- and TLR4-driven inflammatory responses via autophagosome-mediated downregulation of the TLR adaptor-transducer MyD88. In vivo, ES-62 protected mice against endotoxic and polymicrobial septic shock by TLR4-mediated induction of autophagy and was protective even when administered after the induction of sepsis. Given that the treatments for septic shock at present are inadequate, the autophagy-dependent mechanism of action by ES-62 might form the basis for urgently needed therapeutic intervention against this life-threatening condition.
Assuntos
Proteínas de Helminto/farmacologia , Fator 88 de Diferenciação Mieloide/metabolismo , Fagossomos/efeitos dos fármacos , Choque Séptico/prevenção & controle , Receptor 4 Toll-Like/metabolismo , Animais , Autofagia/efeitos dos fármacos , Autofagia/imunologia , Células Cultivadas , Feminino , Humanos , Immunoblotting , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Microscopia Eletrônica , Fator 88 de Diferenciação Mieloide/imunologia , Fagossomos/imunologia , Fagossomos/metabolismo , Choque Séptico/genética , Choque Séptico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Receptor 2 Toll-Like/imunologia , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologiaRESUMO
Atopic allergy is characterized by an increase in IgE antibodies that signal through the high-affinity Fcepsilon receptor (FcepsilonRI) to induce the release of inflammatory mediators from mast cells. For unknown reasons, the prevalence of allergic diseases has recently increased steeply in the developed world. However, this increase has not been mirrored in developing countries, even though IgE concentrations are often greatly elevated in individuals from these countries, owing to nonspecific IgE induction by universally present parasitic worms. Here we offer one explanation for this paradox based on the properties of ES-62, a molecule secreted by filarial nematodes. We found that highly purified, endotoxin-free ES-62 directly inhibits the FcepsilonRI-induced release of allergy mediators from human mast cells by selectively blocking key signal transduction events, including phospholipase D-coupled, sphingosine kinase-mediated calcium mobilization and nuclear factor-kappaB activation. ES-62 mediates these effects by forming a complex with Toll-like receptor 4, which results in the sequestration of protein kinase C-alpha (PKC-alpha). This causes caveolae/lipid raft-mediated, proteasome-independent degradation of PKC-alpha, a molecule important for the coupling of FcepsilonRI to phospholipase D and mast cell activation. We also show that ES-62 is able to protect mice from mast cell-dependent hypersensitivity in the skin and lungs, indicating that it has potential as a novel therapeutic for allergy.
Assuntos
Filarioidea/imunologia , Proteínas de Helminto/fisiologia , Mastócitos/imunologia , Mastócitos/metabolismo , Receptores de IgE/antagonistas & inibidores , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/parasitologia , Cálcio/antagonistas & inibidores , Cálcio/metabolismo , Degranulação Celular/imunologia , Linhagem Celular , Células Cultivadas , Regulação para Baixo/imunologia , Feminino , Humanos , Mastócitos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Proteína Quinase C-alfa/antagonistas & inibidores , Proteína Quinase C-alfa/biossíntese , Proteína Quinase C-alfa/genética , Ratos , Receptores de IgE/fisiologia , Transdução de Sinais/imunologiaRESUMO
Nitric oxide is implicated in the pathogenesis of various neuropathologies characterized by oxidative stress. Although nitric oxide has been reported to be involved in the exacerbation of oxidative stress observed in several neuropathologies, existent data fail to provide a holistic description of how nitrergic pathobiology elicits neuronal injury. Here we provide a comprehensive description of mechanisms contributing to nitric oxide induced neuronal injury by global transcriptomic profiling. Microarray analyses were undertaken on RNA from murine primary cortical neurons treated with the nitric oxide generator DETA-NONOate (NOC-18, 0.5 mM) for 8-24 hrs. Biological pathway analysis focused upon 3672 gene probes which demonstrated at least a ±1.5-fold expression in a minimum of one out of three time-points and passed statistical analysis (one-way anova, P < 0.05). Numerous enriched processes potentially determining nitric oxide mediated neuronal injury were identified from the transcriptomic profile: cell death, developmental growth and survival, cell cycle, calcium ion homeostasis, endoplasmic reticulum stress, oxidative stress, mitochondrial homeostasis, ubiquitin-mediated proteolysis, and GSH and nitric oxide metabolism. Our detailed time-course study of nitric oxide induced neuronal injury allowed us to provide the first time a holistic description of the temporal sequence of cellular events contributing to nitrergic injury. These data form a foundation for the development of screening platforms and define targets for intervention in nitric oxide neuropathologies where nitric oxide mediated injury is causative.
Assuntos
Apoptose/fisiologia , Perfilação da Expressão Gênica , Neurônios/patologia , Neurônios/fisiologia , Óxido Nítrico/metabolismo , Transdução de Sinais/fisiologia , Transcriptoma , Animais , Sobrevivência Celular , Células Cultivadas , Biologia Computacional , Regulação da Expressão Gênica , Camundongos , Dados de Sequência Molecular , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo/fisiologia , Fatores de TempoRESUMO
The proinflammatory cytokine IL-17A is considered a crucial player in rheumatoid arthritis (RA) pathogenesis. In experimental models of autoimmune arthritis, it has been suggested that the cellular source of IL-17A is CD4(+) T cells (Th17 cells). However, little is known about the source of IL-17 in human inflamed RA tissue. We explored the cellular sources of IL-17A in human RA synovium. Surprisingly, only a small proportion of IL-17-expressing cells were T cells, and these were CCR6 negative. Unexpectedly, the majority of IL-17A expression colocalized within mast cells. Furthermore, we demonstrated in vitro that mast cells produced RORC-dependent IL-17A upon stimulation with TNF-alpha, IgG complexes, C5a, and LPS. These data are consistent with a crucial role for IL-17A in RA pathogenesis but suggest that in addition to T cells innate immune pathways particularly mediated via mast cells may be an important component of the effector IL-17A response.
Assuntos
Artrite Reumatoide/imunologia , Interleucina-17/imunologia , Mastócitos/imunologia , Membrana Sinovial/imunologia , Artrite Reumatoide/metabolismo , Humanos , Imuno-Histoquímica , Interleucina-17/biossíntese , Mastócitos/metabolismo , Microscopia de Fluorescência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Membrana Sinovial/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Rheumatoid arthritis pathogenesis comprises dysregulation in both innate and adaptive immunity. There is therefore intense interest in the factors that integrate these immunologic pathways in rheumatoid arthritis. In this paper, we report that IL-33, a novel member of the IL-1 family, can exacerbate anti-glucose-6-phosphate isomerase autoantibody-induced arthritis (AIA). Mice lacking ST2 (ST2(-/-)), the IL-33 receptor alpha-chain, developed attenuated AIA and reduced expression of articular proinflammatory cytokines. Conversely, treatment of wild-type mice with rIL-33 significantly exacerbated AIA and markedly enhanced proinflammatory cytokine production. However, IL-33 failed to increase the severity of the disease in mast cell-deficient or ST2(-/-) mice. Furthermore, mast cells from wild-type, but not ST2(-/-), mice restored the ability of ST2(-/-) recipients to mount an IL-33-mediated exacerbation of AIA. IL-33 also enhanced autoantibody-mediated mast cell degranulation in vitro and in synovial tissue in vivo. Together these results demonstrate that IL-33 can enhance autoantibody-mediated articular inflammation via promoting mast cell degranulation and proinflammatory cytokine production. Because IL-33 is derived predominantly from synovial fibroblasts, this finding provides a novel mechanism whereby a host tissue-derived cytokine can regulate effector adaptive immune response via enhancing innate cellular activation in inflammatory arthritis.
Assuntos
Artrite Experimental/imunologia , Autoanticorpos/imunologia , Interleucinas/toxicidade , Receptores de Interleucina/deficiência , Animais , Artrite Experimental/induzido quimicamente , Artrite Experimental/genética , Degranulação Celular/efeitos dos fármacos , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Glucose-6-Fosfato Isomerase/imunologia , Proteína 1 Semelhante a Receptor de Interleucina-1 , Interleucina-33 , Interleucinas/genética , Articulações/efeitos dos fármacos , Articulações/metabolismo , Articulações/patologia , Mastócitos/imunologia , Mastócitos/metabolismo , Mastócitos/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Receptores de Interleucina/genéticaRESUMO
Anaphylactic shock is characterized by elevated immunoglobulin-E (IgE) antibodies that signal via the high affinity Fc epsilon receptor (Fc epsilonRI) to release inflammatory mediators. Here we report that the novel cytokine interleukin-33 (IL-33) potently induces anaphylactic shock in mice and is associated with the symptom in humans. IL-33 is a new member of the IL-1 family and the ligand for the orphan receptor ST2. In humans, the levels of IL-33 are substantially elevated in the blood of atopic patients during anaphylactic shock, and in inflamed skin tissue of atopic dermatitis patients. In murine experimental atopic models, IL-33 induced antigen-independent passive cutaneous and systemic anaphylaxis, in a T cell-independent, mast cell-dependent manner. In vitro, IL-33 directly induced degranulation, strong eicosanoid and cytokine production in IgE-sensitized mast cells. The molecular mechanisms triggering these responses include the activation of phospholipase D1 and sphingosine kinase1 to mediate calcium mobilization, Nuclear factor-kappaB activation, cytokine and eicosanoid secretion, and degranulation. This report therefore reveals a hitherto unrecognized pathophysiological role of IL-33 and suggests that IL-33 may be a potential therapeutic target for anaphylaxis, a disease of considerable unmet medical need.
Assuntos
Anafilaxia/imunologia , Interleucinas/fisiologia , Animais , Cálcio/metabolismo , Degranulação Celular , Quimiocinas/biossíntese , Citocinas/biossíntese , Dermatite/imunologia , Eicosanoides/biossíntese , Feminino , Humanos , Imunoglobulina E/imunologia , Interleucina-33 , Masculino , Mastócitos/citologia , Camundongos , NF-kappa B/biossínteseRESUMO
Inhibition of proteasome degradation pathway has been implicated in neuronal cell death leading to neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. We and others demonstrated that treatment of cortical neurons with the proteasomal inhibitor lactacystin leads to apoptosis. We discovered by microarray analysis that lactacystin treatment modulates the expression of both potentially neuroprotective as well as pro-apoptotic genes in neurons. However, the significance of the genes which upon transcriptional modulation contributed to proteasomal inhibition-induced apoptosis, remained unidentified. By employing microarray analysis to decipher the time-dependent changes in transcription of these genes in cultured cortical neurons, we discovered different groups of genes were transcriptionally regulated in the early and late phase of lactacystin-induced cell death. In the early phase, several neuroprotective genes such as those encoding the proteasome subunits and ubiquitin-associated enzymes, as well as the heat-shock proteins (HSP) were up-regulated. However, the pro-apoptotic endoplasmic reticulum (ER) stress-associated genes were also up-regulated at the early phase of lactacystin-induced neuronal cell death. In the late phase, genes encoding antioxidants and calcium-binding proteins were up-regulated while those associated with cholesterol biosynthesis were down-regulated. The data suggest that ER stress may participate in mediating the apoptotic responses induced by proteasomal inhibition. The up-regulation of the neuroprotective antioxidant genes and calcium-binding protein genes and down-regulation of the cholesterol biosynthesis genes in the later phase are likely consequences of stimulation of the pro-apoptotic signaling pathways in the early phase of lactacystin treatment.
Assuntos
Acetilcisteína/análogos & derivados , Córtex Cerebral/citologia , Inibidores de Cisteína Proteinase/farmacologia , Retículo Endoplasmático/metabolismo , Neurônios , Inibidores de Proteassoma , Estresse Fisiológico/genética , Acetilcisteína/farmacologia , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Perfilação da Expressão Gênica , Camundongos , Análise em Microsséries , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Regulação para CimaRESUMO
Recently the role of hydrogen sulphide (H(2) S) as a gasotransmitter stimulated wide interest owing to its involvement in Alzheimer's disease and ischemic stroke. Previously we demonstrated the importance of functional ionotropic glutamate receptors (GluRs) by neurons is critical for H(2) S-mediated dose- and time-dependent injury. Moreover N-methyl-D-aspartate receptor (NMDAR) antagonists abolished the consequences of H(2) S-induced neuronal death. This study focuses on deciphering the downstream effects activation of NMDAR on H(2) S-mediated neuronal injury by analyzing the time-course of global gene profiling (5, 15, and 24 h) to provide a comprehensive description of the recruitment of NMDAR-mediated signaling. Microarray analyses were performed on RNA from cultured mouse primary cortical neurons treated with 200 µM sodium hydrosulphide (NaHS) or NMDA over a time-course of 5-24 h. Data were validated via real-time PCR, western blotting, and global proteomic analysis. A substantial overlap of 1649 genes, accounting for over 80% of NMDA global gene profile present in that of H(2) S and over 50% vice versa, was observed. Within these commonly occurring genes, the percentage of transcriptional consistency at each time-point ranged from 81 to 97%. Gene families involved included those related to cell death, endoplasmic reticulum stress, calcium homeostasis, cell cycle, heat shock proteins, and chaperones. Examination of genes exclusive to H(2) S-mediated injury (43%) revealed extensive dysfunction of the ubiquitin-proteasome system. These data form a foundation for the development of screening platforms and define targets for intervention in H(2) S neuropathologies where NMDAR-activated signaling cascades played a substantial role.
Assuntos
Córtex Cerebral/efeitos dos fármacos , Agonistas de Aminoácidos Excitatórios/farmacologia , Perfilação da Expressão Gênica , Neurônios/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Sulfetos/farmacologia , Animais , Western Blotting , Morte Celular , Sobrevivência Celular , Células Cultivadas , Córtex Cerebral/embriologia , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Relação Dose-Resposta a Droga , Perfilação da Expressão Gênica/métodos , Camundongos , N-Metilaspartato/farmacologia , Neurônios/metabolismo , Neurônios/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteômica/métodos , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Fatores de TempoRESUMO
Receptors for the fragment crystallizable region of immunoglobulin-G (FcgammaRs) play an important role in linking the humoral and cellular arms of the immune response. In this study, we present a comprehensive functional comparison of 2 human Fc-receptors, FcgammaRI and FcgammaRIIa. Activation of FcgammaRI results in a novel signaling cascade that links phospholipase D1 to sphingosine kinase-1 in U937 cells and primary human monocytes. This induces the expression of proinflammatory mediators and is associated with trafficking of immune complexes into human leukocyte antigen-DM positive antigen-processing compartments coupled with improved MHC class II-mediated antigen presentation to T lymphocytes. In contrast, activation of FcgammaRIIa elicits signaling through phospholipase Cgamma1, resulting in increases in intracellular calcium, activation of nicotinamide adenine dinucleotide phosphate-oxidative burst, and differential membrane trafficking combined with impaired antigen presentation and proinflammatory cytokine expression. These data provide a mechanistic insight into the disparate activities associated with Fc receptors in immunity, namely, reinforcement of immune responses through stimulation of proinflammatory signaling and antigen presentation, versus the maintenance of immunologic homeostasis through the noninflammatory clearance of immune complexes.
Assuntos
Sinalização do Cálcio , Membrana Celular/imunologia , Membrana Celular/metabolismo , Receptores de IgG/imunologia , Receptores de IgG/metabolismo , Apresentação de Antígeno/imunologia , Células Cultivadas , Citocinas/metabolismo , Regulação da Expressão Gênica , Humanos , Isoenzimas/metabolismo , Estresse Oxidativo , Fosfolipase C gama/metabolismo , Ligação Proteica , Proteína Quinase C/metabolismo , Transporte Proteico , Receptores de IgG/genéticaRESUMO
VAMP8, a member of the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) family of fusion proteins, initially characterized in endosomal and endosomal-lysosomal fusion, may also function in regulated exocytosis. VAMP8 physiological function in inflammation has not been elucidated. In this paper, we show that deficiency of VAMP8 protects mice from anaphylatoxin (C5a)-induced neutropenia, peritonitis, and systemic inflammation. We show that, in vivo, VAMP8 deletion inhibits neutropenia and phagocyte recruitment. We also show that in macrophages, VAMP8 localizes on secretory granules and degranulation is inhibited in VAMP8-deficient macrophages. Moreover, VAMP8(-/-) mice show reduced systemic inflammation with inhibition of serum TNF-alpha levels, whereas IL-1beta, IL-6, and MIP1alpha release are not affected. In wild-type macrophages, TNF-alpha colocalizes with VAMP8-positive vesicles, and in VAMP8-deficient macrophages, the TNF-alpha release is inhibited. Furthermore, VAMP8 regulates the release of TNF-alpha and beta-hexosaminidase triggered by fMLP, and VAMP8(-/-) mice are protected from fMLP-induced peritonitis. These data demonstrate that the VAMP8 vesicle-associated-SNARE is required for the proper trafficking of secretory lysosomal granules for exocytosis in macrophages and for the release of the potent proinflammatory cytokine, TNF-alpha.
Assuntos
Anafilatoxinas/farmacologia , Degranulação Celular/efeitos dos fármacos , Proteínas R-SNARE/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Citocinas/sangue , Exocitose , Fatores Imunológicos , Inflamação , Macrófagos , Camundongos , Camundongos Knockout , N-Formilmetionina Leucil-Fenilalanina/toxicidade , Neutropenia , Peritonite/induzido quimicamente , Fagócitos , Proteínas R-SNARE/deficiência , Vesículas SecretóriasRESUMO
Mast cell degranulation is pivotal to allergic diseases; investigating novel pathways triggering mast cell degranulation would undoubtedly have important therapeutic potential. FcepsilonRI-mediated degranulation has contradictorily been shown to require SphK1 or SphK2, depending on the reports. We investigated the in vitro and in vivo specific role(s) of SphK1 and SphK2 in FcepsilonRI-mediated responses, using specific small interfering RNA-gene silencing. The small interfering RNA-knockdown of SphK1 in mast cells inhibited several signaling mechanisms and effector functions, triggered by FcepsilonRI stimulation including: Ca(2+) signals, NFkappaB activation, degranulation, cytokine/chemokine, and eicosanoid production, whereas silencing SphK2 had no effect at all. Moreover, silencing SPHK1 in vivo, in different strains of mice, strongly inhibited mast cell-mediated anaphylaxis, including inhibition of vascular permeability, tissue mast cell degranulation, changes in temperature, and serum histamine and cytokine levels, whereas silencing SPHK2 had no effect and the mice developed anaphylaxis. Our data differ from a recent report using SPHK1(-/-) and SPHK2(-/-) mice, which showed that SphK2 was required for FcepsilonRI-mediated mast cell responses. We performed experiments in mast cells derived from SPHK1(-/-) and SPHK2(-/-) mice and show that the calcium response and degranulation, triggered by FcepsilonRI-cross-linking, is not different from that triggered in wild-type cells. Moreover, IgE-mediated anaphylaxis in the knockout mice showed similar levels in temperature changes and serum histamine to that from wild-type mice, indicating that there was no protection from anaphylaxis for either knockout mice. Thus, our data strongly suggest a previously unrecognized compensatory mechanism in the knockout mice, and establishes a role for SphK1 in IgE-mediated mast cell responses.
Assuntos
Mastócitos/enzimologia , Mastócitos/imunologia , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Receptores de IgE/fisiologia , Transdução de Sinais/imunologia , Anafilaxia/genética , Anafilaxia/imunologia , Anafilaxia/prevenção & controle , Animais , Degranulação Celular/imunologia , Células Cultivadas , Reagentes de Ligações Cruzadas/metabolismo , Inativação Gênica , Isoenzimas/deficiência , Isoenzimas/genética , Isoenzimas/fisiologia , Masculino , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfotransferases (Aceptor do Grupo Álcool)/deficiência , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Transporte Proteico/imunologia , Interferência de RNA , Receptores de IgE/metabolismo , Transdução de Sinais/genéticaRESUMO
Sphingosine kinase (SphK) phosphorylates sphingosine into sphingosine-1-phosphate (S1P). S1P plays a critical role in angiogenesis, inflammation, and various pathologic conditions. To date, two mammalian isoenzymes, SphK1 and SphK2, have been identified. Although both SphK1 and SphK2 share overall homology and produce the common product, S1P, it has been proposed they display different unique and separate functions. In this study, we examined the role of SphK1 and SphK2 in a murine collagen-induced arthritis model by down-regulating each isoenzyme via specific small interfering RNA (siRNA). Prophylactic i.p. administration of SphK1 siRNA significantly reduced the incidence, disease severity, and articular inflammation compared with control siRNA recipients. Treatment of SphK1 siRNA also down-regulated serum levels of S1P, IL-6, TNF-alpha, IFN-gamma, and IgG2a anti-collagen Ab. Ex vivo analysis demonstrated significant suppression of collagen-specific proinflammatory/Th1 cytokine (IL-6, TNF-alpha, IFN-gamma) release in SphK siRNA-treated mice. Interestingly, mice received with SphK2 siRNA develop more aggressive disease; higher serum levels of IL-6, TNF-alpha, and IFN-gamma; and proinflammatory cytokine production to collagen in vitro when compared with control siRNA recipients. Together, these results demonstrate the distinct immunomodulatory roles of SphK1 and SphK2 in the development of inflammatory arthritis by regulating the release of proinflammatory cytokines and T cell responses. These findings raise the possibility that drugs which specifically target SphK1 activity may play a beneficial role in the treatment of inflammatory arthritis.
Assuntos
Artrite Experimental/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Animais , Artrite Experimental/imunologia , Citocinas/sangue , Inflamação/enzimologia , Interferon gama/sangue , Interleucina-6/sangue , Isoenzimas/fisiologia , Camundongos , RNA Interferente Pequeno/farmacologia , Células Th1/imunologia , Fator de Necrose Tumoral alfa/sangueRESUMO
Deletion of mouse preprotachykinin-A (PPTA), which encodes mainly for neuropeptide substance P, has been shown to protect against lung injury and mortality in sepsis. This study explored microarray-based differential gene expression profiles in mouse lung tissue 8 h after inducing microbial sepsis and the effect of PPTA gene deletion. A range of genes differentially expressed (more than two-fold) in microarray analysis was assessed, comparing wild-type and PPTA-knockout septic mice with their respective sham controls, and the data were further validated. Genetic deletion of substance P resulted in a significantly different expression profile of genes involved in inflammation and immunomodulation after the induction of sepsis, compared with wild-type mice. Interestingly, apart from the various proinflammatory mediators, the antiinflammatory cytokine interleukin-1 receptor antagonist gene (IL1RN) was also elevated much more in PPTA(-/-) septic mice. In addition, semiquantitative RT-PCR analysis supported the microarray data. The microarray data imply that the elevated levels of inflammatory gene expression in the early stages of sepsis in PPTA-knockout mice are possibly aimed to resolve the infection without excessive immunosuppression. As scientists are divided over the effects of pro- and antiinflammatory mediators in sepsis, it seems prudent to define the status depending on a complete genome profile. This is the first report exploring pulmonary gene expression profiles using microarray analysis in PPTA-knockout mice subjected to cecal ligation and puncture-induced sepsis and providing additional biological insight into the protection received against lung injury and mortality.
Assuntos
Bacteriemia/metabolismo , Pneumopatias/metabolismo , Precursores de Proteínas/deficiência , Taquicininas/deficiência , Análise de Variância , Animais , Bacteriemia/genética , Bacteriemia/microbiologia , Quimiocinas/genética , Quimiocinas/metabolismo , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Inflamação/genética , Inflamação/metabolismo , Proteína Antagonista do Receptor de Interleucina 1/genética , Proteína Antagonista do Receptor de Interleucina 1/metabolismo , Pneumopatias/genética , Pneumopatias/microbiologia , Camundongos , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Taquicininas/genética , Taquicininas/metabolismoRESUMO
The anti-inflammatory activity of the phytoalexin resveratrol (RSV) was evaluated in C5 anaphylatoxin (C5a)-stimulated primary neutrophils and in a mouse model of acute peritonitis. Pretreatment of human and mouse neutrophils with RSV significantly blocked oxidative burst, leukocyte migration, degranulation, and inflammatory cytokine production. The anti-inflammatory activity of RSV was a function of inhibition of sphingosine kinase (SphK) activity (IC(50) approximately 20 microM) within 5 min of exposure, its membrane localization, and SphK1-mediated Ca(2+) release. As an experimental control, the SphK1 pharmacological inhibitor N,N-dimethyl sphingosine (DMS) was used to compare the inhibitory effect of RSV. We also provide evidence that the SphK inhibitory effect of RSV was mediated via its ability to block phospholipase D (PLD) activity and membrane recruitment. Furthermore, RSV blocked ERK1/2 phosphorylation, which functioned independently of SphK1 in this study. To provide in vivo relevance to these data, C5a-induced model of acute peritonitis was established, and the effects of prior injection of RSV were investigated. Indeed, prior injection of RSV virtually completely attenuated the effects of C5a on vascular permeability, neutrophil migration, release of interleukin 1beta, tumor necrosis factor alpha, interleukin 6, and the chemokine MIP-1alpha. Taken together, these data demonstrate strong anti-inflammatory activity of RSV in vitro and in vivo and highlight SphK1 as a potential target of this remarkable phytoalexin. These data could have tremendous implications for the clinical use of RSV in inflammatory pathologies.
Assuntos
Complemento C5a/administração & dosagem , Inflamação/prevenção & controle , Fosfolipase D/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Estilbenos/administração & dosagem , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Transporte Biológico Ativo/efeitos dos fármacos , Degranulação Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Quimiocinas/metabolismo , Quimiotaxia de Leucócito/efeitos dos fármacos , Citocinas/metabolismo , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Técnicas In Vitro , Inflamação/enzimologia , Inflamação/etiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/fisiologia , Peritonite/etiologia , Peritonite/fisiopatologia , Peritonite/prevenção & controle , Explosão Respiratória/efeitos dos fármacos , ResveratrolRESUMO
The mitochondrial respiratory chain (MRC) plays crucial roles in cellular energy production. However, its function in early embryonic development remains largely unknown. To address this issue, GRIM-19, a newly identified MRC complex I subunit, was knocked down in Xenopus laevis embryos. A severe deficiency in heart formation was observed, and the deficiency could be rescued by reintroducing human GRIM-19 mRNA. The mechanism involved was further investigated. We found that the activity of NFAT, a transcription factor family that contributes to early organ development, was downregulated in GRIM-19 knockdown embryos. Furthermore, the expression of a constitutively active form of mouse NFATc4 in these embryos rescued the heart developmental defects. NFAT activity is controlled by a calcium-dependent protein phosphatase, calcineurin, which suggests that calcium signaling may be disrupted by GRIM-19 knockdown. Indeed, both the calcium response and calcium-induced NFAT activity were impaired in the GRIM-19 or NDUFS3 (another complex I subunit) knockdown cell lines. We also showed that NFAT can rescue expression of Nkx2.5, which is one of the key genes for early heart development. Our data demonstrated the essential role of MRC in heart formation and revealed the signal transduction and gene expression cascade involved in this process.
Assuntos
Sinalização do Cálcio , Coração/embriologia , Mitocôndrias/fisiologia , Fatores de Transcrição NFATC/metabolismo , Xenopus laevis/embriologia , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose/biossíntese , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/genética , Neoplasias da Mama/patologia , Calcineurina/metabolismo , Cálcio/análise , Linhagem Celular Tumoral , DNA Complementar , Transporte de Elétrons , Embrião não Mamífero , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Células HeLa , Humanos , Hibridização In Situ , Células Jurkat , Luciferases/metabolismo , Dados de Sequência Molecular , Músculo Esquelético/ultraestrutura , Miocárdio/ultraestrutura , NADH NADPH Oxirredutases/biossíntese , NADH NADPH Oxirredutases/química , NADH NADPH Oxirredutases/genética , Fatores de Transcrição NFATC/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Homologia de Sequência de Aminoácidos , Transcrição Gênica , TransfecçãoRESUMO
Sphingosine kinase (SphK) is a key enzyme in the sphingolipid metabolic pathway responsible for phosphorylating sphingosine into sphingosine-1-phosphate (S1P). SphK/S1P play a critical role in angiogenesis, inflammation, and various pathologic conditions. Recently, S1P(1) receptor was found to be expressed in rheumatoid arthritis (RA) synovium, and S1P signaling via S1P(1) enhances synoviocyte proliferation, COX-2 expression, and prostaglandin E(2) production. Here, we examined the role of SphK/S1P in RA using a potent SphK inhibitor, N,N-dimethylsphingosine (DMS), and a molecular approach against one of its isoenzymes, SphK1. We observed that levels of S1P in the synovial fluid of RA patients were significantly higher than those of osteoarthritis patients. Additionally, DMS significantly reduced the levels of TNF-alpha, IL-6, IL-1beta, MCP-1, and MMP-9 in cell-contact assays using both Jurkat-U937 cells and RA PBMCs. In a murine collagen-induced arthritis model, i.p. administration of DMS significantly inhibited disease severity and reduced articular inflammation and joint destruction. Treatment of DMS also down-regulated serum levels IL-6, TNF-alpha, IFN-gamma, S1P, and IgG1 and IgG2a anti-collagen Ab. Furthermore, DMS-treated mice also displayed suppressed proinflammatory cytokine production in response to type II collagen in vitro. Moreover, similar reduction in incidence and disease activity was observed in mice treated with SphK1 knock-down via small interfering RNA approach. Together, these results demonstrate SphK modulation may provide a novel approach in treating chronic autoimmune conditions such as RA by inhibiting the release of pro-inflammatory cytokines.
Assuntos
Artrite Reumatoide/imunologia , Leucócitos Mononucleares/imunologia , Lisofosfolipídeos/imunologia , Fosfotransferases (Aceptor do Grupo Álcool)/imunologia , Transdução de Sinais/imunologia , Esfingosina/análogos & derivados , Líquido Sinovial/imunologia , Animais , Artrite Reumatoide/enzimologia , Artrite Reumatoide/patologia , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo II/imunologia , Colágeno Tipo II/farmacologia , Ciclo-Oxigenase 2/biossíntese , Ciclo-Oxigenase 2/metabolismo , Citocinas/sangue , Citocinas/imunologia , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/imunologia , Humanos , Inflamação/enzimologia , Inflamação/imunologia , Inflamação/patologia , Mediadores da Inflamação/sangue , Mediadores da Inflamação/imunologia , Células Jurkat , Leucócitos Mononucleares/enzimologia , Leucócitos Mononucleares/patologia , Lisofosfolipídeos/metabolismo , Metaloproteinase 9 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/imunologia , Camundongos , Camundongos Endogâmicos DBA , Neovascularização Fisiológica/imunologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , RNA Interferente Pequeno/imunologia , Receptores de Lisoesfingolipídeo/biossíntese , Receptores de Lisoesfingolipídeo/imunologia , Transdução de Sinais/efeitos dos fármacos , Esfingosina/imunologia , Esfingosina/metabolismo , Esfingosina/farmacologia , Líquido Sinovial/enzimologia , Células U937RESUMO
During the last few years, it has become clear that sphingolipids are sources of important signalling molecules. Particularly, the sphingolipid metabolites, ceramide and S1P, have emerged as a new class of potent bioactive molecules, implicated in a variety of cellular processes such as cell differentiation, apoptosis, and proliferation. Sphingomyelin (SM) is the major membrane sphingolipid and is the precursor for the bioactive products. Ceramide is formed from SM by the action of sphingomyelinases (SMase), however, ceramide can be very rapidly hydrolysed, by ceramidases to yield sphingosine, and sphingosine can be phosphorylated by sphingosine kinase (SphK) to yield S1P. In immune cells, the sphingolipid metabolism is tightly related to the main stages of immune cell development, differentiation, activation, and proliferation, transduced into physiological responses such as survival, calcium mobilization, cytoskeletal reorganization and chemotaxis. Several biological effectors have been shown to promote the synthesis of S1P, including growth factors, cytokines, and antigen and G-protein-coupled receptor agonists. Interest in S1P focused recently on two distinct cellular actions of this lipid, namely its function as an intracellular second messenger, capable of triggering calcium release from internal stores, and as an extracellular ligand activating specific G protein-coupled receptors. Inhibition of SphK stimulation strongly reduced or even prevented cellular events triggered by several proinflammatory agonists, such as receptor-stimulated DNA synthesis, Ca(2+) mobilization, degranulation, chemotaxis and cytokine production. Another very important observation is the direct role played by S1P in chemotaxis, and cellular escape from apoptosis. As an extracellular mediator, several studies have now shown that S1P binds a number of G-protein-coupled receptors (GPCR) encoded by endothelial differentiation genes (EDG), collectively known as the S1P-receptors. Binding of S1P to these receptors trigger an wide range of cellular responses including proliferation, enhanced extracellular matrix assembly, stimulation of adherent junctions, formation of actin stress fibres, and inhibition of apoptosis induced by either ceramide or growth factor withdrawal. Moreover, blocking S1P1-receptor inhibits lymphocyte egress from lymphatic organs. This review summarises the evidence linking SphK signalling pathway to immune-cell activation and based on these data discuss the potential for targeting SphKs to suppress inflammation and other pathological conditions.
Assuntos
Ceramidas/metabolismo , Sistema Imunitário/enzimologia , Linfócitos/enzimologia , Linfócitos/imunologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Transdução de Sinais , Esfingomielina Fosfodiesterase/metabolismo , Esfingomielinas/metabolismo , Animais , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Sinalização do Cálcio , Moléculas de Adesão Celular/metabolismo , Humanos , Hipersensibilidade/tratamento farmacológico , Hipersensibilidade/imunologia , Hipersensibilidade/metabolismo , Sistema Imunitário/citologia , Sistema Imunitário/imunologia , Inflamação/tratamento farmacológico , Inflamação/imunologia , Inflamação/metabolismo , Linfócitos/metabolismo , Lisofosfolipídeos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Esfingomielinas/imunologia , Esfingosina/análogos & derivados , Esfingosina/metabolismoRESUMO
Telomeres and telomerase appear to participate in the repair of broken DNA ends produced by oxidative damage. Arsenite is an environmental contaminant and a potent human carcinogen, which induces oxidative stress on cells via the generation of reactive oxygen species affecting cell viability and chromosome stability. It promotes telomere attrition and reduces cell survival by apoptosis. In this study, we used mouse embryonic fibroblasts (MEFs) from mice lacking telomerase RNA component (mTERC(-/-) mice) with long (early passage or EP) and short (late passage or LP) telomeres to investigate the extent of oxidative damage by comparing the differences in DNA damage, chromosome instability, and cell survival at 24 and 48 h of exposure to sodium arsenite (As3+; NaAsO2). There was significantly high level of DNA damage in mTERC(-/-) cells with short telomeres as determined by alkaline comet assay. Consistent with elevated DNA damage, increased micronuclei (MN) induction reflecting gross genomic instability was also observed. Fluorescence in situ hybridization (FISH) analysis revealed that increasing doses of arsenite augmented the chromosome aberrations, which contributes to genomic instability leading to possibly apoptotic cell death and cell cycle arrest. Microarray analysis has revealed that As3+ treatment altered the expression of 456 genes of which 20% of them have known functions in cell cycle and DNA damage signaling and response, cell growth, and/or maintenance. Results from our studies imply that short dysfunctional telomeres impair the repair of oxidative damage caused by arsenite. The results will have implications in risk estimation as well as cancer chemotherapy.