RESUMO
Guanine-nucleotide exchange factors (GEFs) stimulate the intrinsic GDP/GTP exchange activity of Ras and promote the formation of active Ras-GTP, which in turn controls diverse signalling networks important for the regulation of cell proliferation, survival, differentiation, vesicular trafficking, and gene expression. RasGEF1b is a GEF, whose expression is induced in macrophages on stimulation with toll-like receptor (TLR) agonists. Here, we showed that in vitro RasGEF1b expression by macrophages is mostly induced by TLR3 (poly I:C) and TLR4 (lipopolysaccharyde) through the MyD88-independent pathway. In vivo infection with the protozoan parasites Trypanosoma cruzi and Plasmodium chabaudi induced RasGEF1b in an MyD88-, TRIF-, and IFN-gamma-dependent manner. Ectopically expressed RasGEF1b was found, mostly, in the heavy membrane fraction of HEK 293T, and by confocal microscopy, it was found to be located at early endosomes. Computational modelling of the RasGEF1b-Ras interaction revealed that RasGEF1b interacts with the binding domain site of Ras, a critical region for interacting with GEFs involved in the activation of Ras-Raf-MEK-ERK pathway. More important, RasGEF1b was found to be closely associated with Ras in live cells and to trigger Ras activity. Altogether, these results indicate that on TLR activation, RasGEF1b may trigger Ras-like proteins and regulate specific biological activities described for this subtype of GTPases.
Assuntos
Endossomos/metabolismo , Receptores Toll-Like/fisiologia , Fatores ras de Troca de Nucleotídeo Guanina/biossíntese , Animais , Células CHO , Cricetinae , Cricetulus , Endossomos/química , Feminino , Células HEK293 , Humanos , Mediadores da Inflamação/metabolismo , Mediadores da Inflamação/fisiologia , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/química , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Toll-Like/metabolismo , Fatores ras de Troca de Nucleotídeo Guanina/metabolismo , Fatores ras de Troca de Nucleotídeo Guanina/fisiologiaRESUMO
Chagas disease (CD) is caused by the infection with the protozoan haemoflagellate Trypanosoma cruzi. This disease is still a great menace to public health, and is largely neglected as it affects mostly the poorest populations of Latin America. Nonetheless, there are neither effective diagnostic markers nor therapeutic options to accurately detect and efficiently cure this chronic infection. In spite of the great advances in the knowledge of the biology of natural transmission, as well as the immunobiology of the host-parasite interaction, the understanding of the pathogenesis of CD remains largely elusive. In the recent decades, a controversy in the research community has developed about the relevance of parasite persistence or autoimmune phenomena in the development of chronic cardiac pathology. One of the most notable aspects of chronic CD is the progressive deterioration of cardiac function, derived mostly from structural derangement, as a consequence of the intense inflammatory process. Here we review the evidence supporting the multifactorial nature of Chagas heart disease comprising pathogen persistence and altered host immunoregulatory mechanisms.
Assuntos
Cardiomiopatia Chagásica/parasitologia , Trypanosoma cruzi/patogenicidade , Animais , Autoimunidade , Cardiomiopatia Chagásica/imunologia , Interações Hospedeiro-Parasita , Humanos , América Latina , Trypanosoma cruzi/fisiologia , VirulênciaRESUMO
Transparent objects (phase objects) are not visible in a standard brightfield optical microscope. In order to see such objects the most used technique is phase-contrast microscopy. In phase-contrast microscopy the contrast observed is proportional to the optical path difference introduced by the object. If the index of refraction is uniform, phase-contrast microscopy then yields a measure of the thickness profile of phase objects. We show that by slightly defocusing an optical microscope operating in brightfield, phase objects become visible. We modeled such an effect and show that the image contrast of a phase object is proportional to the amount of defocusing and proportional to the two-dimensional Laplacian of the optical path difference introduced by the object. For uniform index of refraction, defocusing microscopy then yields a measure of the curvature profile of phase objects. We extended our previous model for thin objects to thick objects. To check our theoretical model, we use as phase objects polystyrene spherical caps and compare their curvature radii obtained by defocusing microscopy (DM) to those obtained with atomic force microscopy (AFM). We also show that for thick curved phase objects one can reconstruct their thickness profiles from DM images. We illustrate the utility of defocusing microscopy in biological systems to study cell motility. In particular, we visualize and quantitatively measure real-time cytoskeleton curvature fluctuations of macrophages (a cell of the innate immune system). The study of such fluctuations might be important for a better understanding of the engulfment process of pathogens during phagocytosis.
Assuntos
Processamento de Imagem Assistida por Computador , Microscopia/métodos , Animais , Calibragem , Movimento Celular , Citoesqueleto/fisiologia , Citoesqueleto/ultraestrutura , Macrófagos/fisiologia , Macrófagos/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Força Atômica , Modelos Teóricos , Óptica e Fotônica , RefratometriaRESUMO
Chromobacterium violaceum is a versatile, Gram-negative beta-protebacterium that grows in a variety of ecosystems in tropical and subtropical areas, such as the water and borders of the Negro River, in the Amazon region of Brazil. Although it is a saprophyte and is generally considered non-pathogenic, sporadic cases of human infection have been described, mainly in young children and in immunodeficient individuals. Although rare, infections with C. violaceum are characterized by rapid dissemination and high mortality. With the complete genome sequence of C. violaceum now available, a detailed description of the molecular arsenal required for this bacterium's remarkable versatility has been revealed. Most importantly, a more detailed picture of its biotechnological properties, including the characteristic violacein pigment, has emerged. The complete genome sequence also enabled us to make a thorough examination of the repertoire of genes encoding probable virulence factors, which determine the potential for pathogenesis. We described a number of genes involved in infectious processes, such as host cell adhesion, [quot ]contact-dependent secretion[quot ] of factors that promote cell invasion, as well as other virulence factors, such as cytolytic proteins. We also described genes involved with the synthesis of lipopolysaccharides and proteoglycan, known to elicit the synthesis of pro-inflammatory cytokines and involved in the detoxification process, which may contribute to the evasion of the bacteria from the host immune response
Assuntos
Chromobacterium/genética , Fatores de Virulência/genética , Genoma Bacteriano , Lipopolissacarídeos/biossíntese , Aderência Bacteriana/genética , Chromobacterium/patogenicidade , Colicinas/biossíntese , Colicinas/genética , Proteínas Hemolisinas/biossíntese , Proteínas Hemolisinas/genética , Indóis , Virulência/genéticaRESUMO
Phase objects can become visible by slightly defocusing an optical microscope, a technique seldom used as a useful tool. We revisited the theory of defocusing and apply it to our optical microscope with optics corrected at infinity. In our approximation, we obtain that the image contrast is proportional to the two-dimensional (2D) Laplacian of the phase difference introduced by the phase object. If the index of refraction of the phase object is uniform the image obtained from defocusing microscopy is the image of curvature (Laplacian of the local thickness) of the phase object, while standard phase-contrast microscopy gives information about the thickness of the object. We made artificial phase objects and measured image contrasts with defocusing microscopy. Measured contrasts are in excellent agreement with our theoretical model. We use defocusing microscopy to study curvature fluctuations (ruffles) on the surface of macrophages (cell of the innate immune system), and try to correlate mechanical properties of macrophage surface and phagocytosis. We observe large coherent propagating structures: Their shape, speed, density are measured and curvature energy estimated. Inhomogeneities of cytoskeleton refractive index, curvature modulations due to thermal fluctuations and/or periodic changes in cytoskeleton-membrane interactions cause random fluctuations in image contrast. From the temporal and spatial contrast correlation functions, we obtain the decay time and correlation length of such fluctuations that are related to their size and the viscoelastic properties of the cytoskeleton. In order to associate the dynamics of cytoskeleton with the process of phagocytosis, we use an optical tweezers to grab a zymosan particle and put it into contact with the macrophage. We then measure the time for a single phagocytosis event. We add the drug cytochalasin D that depolymerizes the cytoskeleton F-actin network: It inhibits the large propagating coherent fluctuations on the cell surface, increases the relaxation time of cytoskeleton fluctuations, and increases the phagocytosis time. Our results suggest that the methods developed in this work can be of utility to assess the importance of cytoskeleton motility in the dynamics of cellular processes such as phagocytosis exhibited by macrophages.
Assuntos
Membrana Celular/patologia , Microscopia/métodos , Actinas/metabolismo , Animais , Membrana Celular/metabolismo , Citocalasina D/farmacologia , Citoesqueleto/metabolismo , Vidro , Processamento de Imagem Assistida por Computador , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Contraste de Fase/métodos , Microscopia de Vídeo , Modelos Estatísticos , Distribuição Normal , Fagocitose , Fatores de Tempo , Zimosan/farmacologiaRESUMO
We demonstrated that 4 mM butyrate induces apoptosis in murine peritoneal macrophages in a dose- and time-dependent manner as indicated by studies of cell viability, flow cytometric analysis of annexin-V binding, DNA ladder pattern and the determination of hypodiploid DNA content. The activity of caspase-3 was enhanced during macrophage apoptosis induced by butyrate and the caspase inhibitor z-VAD-FMK (100 microM) inhibited the butyrate effect, indicating the major role of the caspase cascade in the process. The levels of butyrate-induced apoptosis in macrophages were enhanced by co-treatment with 1 microg/ml bacterial lipopolysaccharide (LPS). However, our data indicate that apoptosis induced by butyrate and LPS involves different mechanisms. Thus, LPS-induced apoptosis was only observed when macrophages were primed with IFN-gamma and was partially dependent on iNOS, TNFR1 and IRF-1 functions as determined in experiments employing macrophages from various knockout mice. In contrast, butyrate-induced macrophage apoptosis was highly independent of IFN-gamma priming and of iNOS, TNFR1 and IRF-1 functions.
Assuntos
Apoptose , Butiratos/farmacologia , Caspases/metabolismo , Macrófagos/efeitos dos fármacos , Óxido Nítrico/biossíntese , Fator de Necrose Tumoral alfa/biossíntese , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Caspase 3 , Inibidores de Caspase , Sobrevivência Celular , Lipopolissacarídeos/farmacologia , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/análise , Peritônio/citologia , Fator de Necrose Tumoral alfa/análiseRESUMO
We demonstrated that 4 mM butyrate induces apoptosis in murine peritoneal macrophages in a dose- and time-dependent manner as indicated by studies of cell viability, flow cytometric analysis of annexin-V binding, DNA ladder pattern and the determination of hypodiploid DNA content. The activity of caspase-3 was enhanced during macrophage apoptosis induced by butyrate and the caspase inhibitor z-VAD-FMK (100 æM) inhibited the butyrate effect, indicating the major role of the caspase cascade in the process. The levels of butyrate-induced apoptosis in macrophages were enhanced by co-treatment with 1 æg/ml bacterial lipopolysaccharide (LPS). However, our data indicate that apoptosis induced by butyrate and LPS involves different mechanisms. Thus, LPS-induced apoptosis was only observed when macrophages were primed with IFN-gamma and was partially dependent on iNOS, TNFR1 and IRF-1 functions as determined in experiments employing macrophages from various knockout mice. In contrast, butyrate-induced macrophage apoptosis was highly independent of IFN-gamma priming and of iNOS, TNFR1 and IRF-1 functions
Assuntos
Animais , Camundongos , Apoptose , Butiratos , Caspases , Macrófagos , Óxido Nítrico , Fator de Necrose Tumoral alfa , Sobrevivência Celular , Lipopolissacarídeos , Camundongos Endogâmicos C57BL , Óxido Nítrico , Peritônio , Fator de Necrose Tumoral alfaRESUMO
The role of cytokines in the control of tissue parasitism and pathogenesis of experimental Chagas' disease was investigated. Wild-type and different cytokine as well as inducible nitric oxide synthase (iNOS) knockout mice were infected with the Colombian strain of Trypanosoma cruzi, and the kinetics of tissue parasitism, inflammatory reaction, parasitemia, and mortality were determined. We demonstrate the pivotal role of the interleukin (IL)-12/interferon (IFN)-gamma/iNOS axis and the antagonistic effect of IL-4 in controlling heart tissue parasitism, inflammation, and host resistance to acute infection with T. cruzi. Further, the heart and central nervous system were shown the main sites of reactivation of T. cruzi infection in mice lacking functional genes for IFN-gamma and IL-12, respectively. Our results also show that in contrast to IFN-gamma knockout (KO) mice, splenocytes from IL-12 KO mice infected with T. cruzi produced low levels of IFN-gamma upon stimulation with antigen. Consistently, high levels of anti-T. cruzi IgG2a antibodies were detected in the sera from IL-12 KO, but not from IFN-gamma KO mice, infected with the Colombian strain of T. cruzi. Thus, our results suggest that the level of IFN-gamma deficiency is a major determinant of the site of reactivation of T. cruzi infection in immunocompromised host.
Assuntos
Doenças do Sistema Nervoso Central/patologia , Doenças do Sistema Nervoso Central/parasitologia , Cardiomiopatia Chagásica/patologia , Cardiomiopatia Chagásica/parasitologia , Doença de Chagas/patologia , Doença de Chagas/parasitologia , Interferon gama/fisiologia , Interleucina-12/fisiologia , Animais , Sistema Nervoso Central/parasitologia , Sistema Nervoso Central/patologia , Suscetibilidade a Doenças , Feminino , Coração/parasitologia , Interferon gama/deficiência , Interferon gama/genética , Interleucina-12/deficiência , Interleucina-12/genética , Interleucina-4/deficiência , Interleucina-4/genética , Interleucina-4/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout/genética , Miocardite/parasitologia , Miocardite/patologia , Miocárdio/patologia , Óxido Nítrico Sintase/deficiência , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/fisiologia , Óxido Nítrico Sintase Tipo II , RecidivaRESUMO
The determinants of the prevalence of CD8(+) T cells in the inflamed myocardium of Trypanosoma cruzi-infected patients and experimental animals are undefined. Using C3H/He mice infected with the Colombiana strain of T. cruzi, we found that the distribution of CD4(+)/CD8(-) and CD4(-)/CD8(+) T cells in the myocardium mirrors the frequency of cells expressing the CD62L(Low)LFA-1(High)VLA-4(High) activation phenotype among CD4(+)/CD8(-) and CD4(-)/CD8(+ )peripheral blood T cells. Consistently, vascular cell adhesion molecule-1-positive endothelial cells and a fine fibronectin network surrounding VLA-4(+) mononuclear cells were found in the inflamed myocardium. Further, interferon gamma (IFN-gamma) and IFN-gamma-induced chemokines (RANTES, MIG and CRG-2/IP-10), as well as JE/MCP-1 and MIP1-alpha, were found to be the dominant cytokines expressed in situ during acute and chronic myocarditis elicited by T. cruzi. In contrast, interleukin 4 mRNA was only detected during the chronic phase. Altogether, the results indicate that the distribution of T-cell subsets in the myocardium of T. cruzi-infected mice reflects the particular profile of adhesion molecules acquired by most peripheral CD8(+) T lymphocytes and point to the possibility that multiple IFN-gamma-inducible molecules present in the inflamed tissue contribute to the establishment and maintenance of T. cruzi-induced myocarditis.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Cardiomiopatia Chagásica/imunologia , Integrinas/análise , Interferon gama/farmacologia , Selectina L/análise , Antígeno-1 Associado à Função Linfocitária/análise , Receptores de Antígenos de Linfócitos T alfa-beta/análise , Receptores de Retorno de Linfócitos/análise , Animais , Moléculas de Adesão Celular/biossíntese , Cardiomiopatia Chagásica/parasitologia , Cardiomiopatia Chagásica/patologia , Quimiocinas/biossíntese , Citocinas/biossíntese , Feminino , Imunofenotipagem , Integrina alfa4beta1 , Camundongos , Camundongos Endogâmicos C3H , Miocárdio/patologia , Parasitemia/mortalidadeRESUMO
A strong activation of macrophages is observed during acute infection with Trypanosoma cruzi. Little is known, however, about the parasite molecules that are responsible for this early activation of innate immunity. Recent studies have shown the stimulatory activity of protozoan-derived glycosylphosphatidylinositol (GPI) anchors on cultured macrophages. In this review, we provide a detailed analysis of the correlation between structure and proinflammatory activity by T. cruzi-derived GPI anchors. We also cover the studies that have identified the Toll-like receptor 2 as a functional GPI receptor and have partially characterized signaling pathways triggered by T. cruzi-derived GPI anchors, which lead to the synthesis of proinflammatory cytokines in macrophages. Finally, we discuss the implications of these findings in resistance and pathogenesis during the infection with T. cruzi.
Assuntos
Doença de Chagas/imunologia , Proteínas de Drosophila , Glicosilfosfatidilinositóis/química , Glicosilfosfatidilinositóis/fisiologia , Trypanosoma cruzi/patogenicidade , Animais , Sequência de Carboidratos , Citocinas/biossíntese , Inflamação/imunologia , Lipopolissacarídeos/química , Macrófagos/imunologia , Glicoproteínas de Membrana/metabolismo , Dados de Sequência Molecular , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade , Receptor 2 Toll-Like , Receptores Toll-LikeRESUMO
Vaccinia virus (VV) triggers a mitogenic signal at an early stage of infection. VV-induced proto-oncogene c-fos mRNA with kinetics paralleling that stimulated by serum. The VV virokine, or vaccinia virus growth factor (VGF), was not crucial for c-fos induction because it was observed upon infection with the virokine-minus mutant VV (VGF(-)). Furthermore, c-fos expression did not require infectious virus particles, as it occurred even with UV-inactivated VV and was equally induced by the different multiplicities of infection, i.e. 1.0, 5.0, and 25.0. c-fos expression was preceded by VV-induced DNA binding activity and was mediated via the cis-acting elements serum response element (SRE), activating protein-1 (AP-1), and cAMP-response element (CRE). VV activated the protein kinases p42MAPK/ERK2 and p44MAPK/ERK1 and the transcription factor ATF1 in a time-dependent manner with kinetics that paralleled those of VV-stimulated DNA-protein complex formation. The mitogenic signal transmission pathways leading to c-fos activation upon VV infection were apparently mediated by the protein kinases MEK, ERK, and PKA. This assumption was based on the findings that: 1) c-fos transcript was down-regulated; 2) the SRE, AP-1, and CRE binding activities were significantly reduced; and 3) the activation of p42MAPK/ERK2, p44MAPK/ERK1, and ATF1 were drastically affected when the viral infections were carried out in the presence of specific protein kinase inhibitor. Moreover, the mutant VV (VGF(-)) was also able to activate ERK1/2. It is noteworthy that virus multiplication was equally affected by the same kinase inhibitors. Taken together, our data provide evidence that the early mitogenic signal triggered upon VV infection relies upon the activation of the protein kinases MEK, ERK, and PKA, which are needed for both signal transduction and virus multiplication.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mitógenos/metabolismo , Vaccinia virus/metabolismo , Vaccinia virus/patogenicidade , Animais , Northern Blotting , Western Blotting , Linhagem Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , DNA/metabolismo , DNA Complementar/metabolismo , Regulação para Baixo , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Cinética , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Endogâmicos BALB C , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Modelos Biológicos , Ligação Proteica , Proteínas Proto-Oncogênicas c-fos/metabolismo , RNA Mensageiro/metabolismo , Elemento de Resposta Sérica/genética , Transdução de Sinais , Fatores de Tempo , Transcrição GênicaRESUMO
Glycosylphosphatidylinositol (GPI) anchors and glycoinositolphospholipids (GIPLs) from parasitic protozoa have been shown to exert a wide variety of effects on cells of the host innate immune system. However, the receptor(s) that are triggered by these protozoan glycolipids has not been identified. Here we present evidence that Trypanosoma cruzi-derived GPI anchors and GIPLs trigger CD25 expression on Chinese hamster ovary-K1 cells transfected with CD14 and Toll-like receptor-2 (TLR-2), but not wild-type (TLR-2-deficient) Chinese hamster ovary cells. The protozoan-derived GPI anchors and GIPLs containing alkylacylglycerol and saturated fatty acid chains or ceramide were found to be active in a concentration range of 100 nM to 1 microM. More importantly, the GPI anchors purified from T. cruzi trypomastigotes, which contain a longer glycan core and unsaturated fatty acids in the sn-2 position of the alkylacylglycerolipid component, triggered TLR-2 at subnanomolar concentrations. We performed experiments with macrophages from TLR-2 knockout and TLR-4 knockout mice, and found that TLR-2 expression appears to be essential for induction of IL-12, TNF-alpha, and NO by GPI anchors derived from T. cruzi trypomastigotes. Thus, highly purified GPI anchors from T. cruzi parasites are potent activators of TLR-2 from both mouse and human origin. The activation of TLR-2 may initiate host innate defense mechanisms and inflammatory response during protozoan infection, and may provide new strategies for immune intervention during protozoan infections.
Assuntos
Proteínas de Drosophila , Glicosilfosfatidilinositóis/fisiologia , Glicoproteínas de Membrana/metabolismo , Receptores de Superfície Celular/metabolismo , Trypanosoma cruzi/imunologia , Animais , Células CHO , Linhagem Celular , Cricetinae , Relação Dose-Resposta Imunológica , Glicolipídeos/fisiologia , Glicosilfosfatidilinositóis/isolamento & purificação , Inflamação/imunologia , Inflamação/parasitologia , Macrófagos/imunologia , Macrófagos/metabolismo , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiologia , Camundongos , Camundongos Knockout , NF-kappa B/fisiologia , Fosfolipídeos/fisiologia , Receptores de Superfície Celular/deficiência , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/fisiologia , Receptores de Interleucina-2/biossíntese , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Receptor 2 Toll-Like , Receptor 4 Toll-Like , Receptores Toll-Like , Transfecção , Trypanosoma cruzi/química , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
The efficacy of the association of paromomycin sulfate (PA) with recombinant (r) interleukin (IL)-12 was investigated by topical treatment of BALB/c mice infected with Leishmania major that displayed fully developed cutaneous lesions. Although healing was observed in PA-treated groups, lesions recurred in 100% of these animals 70 days after treatment. In contrast, lesions were absent in a high proportion of PA- and rIL-12-treated mice 120 days after treatment. The PA/rIL-12-treated mice had a switch in cytokine response, from high IL-4 and low interferon (IFN)-gamma levels to low IL-4 and high IFN-gamma levels, and reductions in parasite load, dissemination of parasites, and inflammation. Thus, the association of rIL-12 to topical chemotherapy for leishmaniasis may be an important strategy for increasing cure rates and decreasing the incidence of relapse.
Assuntos
Antibacterianos/uso terapêutico , Antiprotozoários/uso terapêutico , Interleucina-12/uso terapêutico , Leishmania major , Leishmaniose/tratamento farmacológico , Paromomicina/uso terapêutico , Administração Cutânea , Animais , Modelos Animais de Doenças , Quimioterapia Combinada , Feminino , Interferon gama/análise , Interleucina-4/análise , Leishmania major/isolamento & purificação , Leishmaniose/imunologia , Leishmaniose/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/análise , Proteínas Recombinantes/uso terapêutico , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Infection with Trypanosoma cruzi causes a strong inflammatory reaction at the inoculation site and, later, in the myocardium. The present study investigates the role of cytokines as modulators of T. cruzi-induced chemokine expression in vivo and in vitro. In macrophage cultures, although the stimulation with interferon (IFN)-gamma increases the expression of IP-10, it blocks KC expression. Tumor necrosis factor (TNF)-alpha, on the other hand, potentiates KC, IP-10, macrophage inflammatory protein-1alpha, and JE/monocyte chemotatic protein-1 expression. Interleukin-10 and transforming growth factor-beta inhibited almost all chemokines tested. The role of IFN-gamma and TNF-alpha in chemokine modulation during infection was investigated in T. cruzi-infected IFN-gamma-deficient (GKO) or TNF-R1/p55-deficient (p55-/-) mice. The expression of chemokines detected in the inoculation site correlated with the infiltrating cell type observed. Although GKO mice had a delayed and intense neutrophilic infiltrate correlating with the expression of KC and macrophage inflammatory protein-2, none of the above was observed in p55-/- mice. The detection of infiltrating T cells, Mig, and IP-10 in the myocardium was observed in wild-type and p55-/-, but not in GKO mice. Together, these results suggest that the regulatory roles of IFN-gamma and TNF-alpha on chemokine expression may play a crucial role in the modulation of the inflammatory response during T. cruzi infection and mediate resistance to infection.
Assuntos
Doença de Chagas/metabolismo , Quimiocinas/biossíntese , Interferon gama/deficiência , Peritonite/parasitologia , Receptores do Fator de Necrose Tumoral/deficiência , Animais , Antígenos CD , Movimento Celular , Doença de Chagas/patologia , Quimiocina CXCL10 , Quimiocina CXCL9 , Quimiocinas/genética , Quimiocinas CXC/metabolismo , Feminino , Imunofenotipagem , Interferon gama/fisiologia , Interleucina-10/fisiologia , Linfócitos/fisiologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , RNA Mensageiro/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral , Fator de Crescimento Transformador beta/fisiologia , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
The ability of up-regulatory [recombinant (r) IFN-gamma, rIFN-beta and rTNF-alpha] and down-regulatory (rIL-4, rIL-10 and rIL-13) cytokines to control the expression of indoleamine 2,3-dioxygenase (INDO) and anti-Toxoplasma activity in the human fibrosarcoma cell line 2C4 was evaluated. Activation of fibroblasts with rIFN-gamma, rIFN-beta and rTNF-alpha resulted in augmentation of INDO expression and activity leading to 40.0, 25.0 and 27.0 % inhibition of tachyzoite growth, respectively. An additive effect was observed when host cells were incubated with rIFN-gamma plus rTNF-alpha. With regard to the down-regulatory cytokines we observed that IL-4 as well as IL-13, but not IL-10, induced significant inhibition of IFN-gamma-induced control of parasite replication, INDO mRNA expression and tryptophan catabolism. Similarly, IL-4 but not IL-10 inhibited the cell surface expression of HLA-DR and CD2 induced by IFN-gamma. Consistent with these findings we were able to detect by reverse transcription-PCR the expression of mRNA for different chains of IL-4 and IL-13 receptors (IL-4Ralpha, IL-13Ralpha1 and IL-13Ralpha2) but not for IL-10 receptor in the 2C4 and other human lung fibroblast cell lines (LL24 and MRC5). Together our results indicate that IL-4 and IL-13, but not IL-10, are implicated in the negative regulation of IFN-gamma-induced anti-Toxoplasma activity in human cells from fibroblast lineage.
Assuntos
Interferon gama/farmacologia , Interleucina-13/farmacologia , Interleucina-4/farmacologia , Toxoplasma/efeitos dos fármacos , Triptofano Oxigenase/biossíntese , Animais , Antígenos CD2/biossíntese , Células Cultivadas , Indução Enzimática/efeitos dos fármacos , Fibroblastos/parasitologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Antígenos HLA-DR/biossíntese , Humanos , Subunidade alfa1 de Receptor de Interleucina-13 , RNA Mensageiro/análise , Receptores de Interleucina/genética , Receptores de Interleucina-10 , Receptores de Interleucina-13 , Receptores de Interleucina-4/genética , Proteínas Recombinantes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Toxoplasma/fisiologia , Triptofano Oxigenase/antagonistas & inibidores , Triptofano Oxigenase/genéticaRESUMO
In the present study, we evaluated the ability of GPI-anchored mucin-like glycoproteins purified from Trypanosoma cruzi trypomastigotes (tGPI-mucin) to trigger phosphorylation of different mitogen-activated protein kinases (MAPKs) and related transcription factors in inflammatory macrophages. Kinetic experiments show that the peak of extracellular signal-related kinase (ERK)-1/ERK-2, stress-activated protein kinase (SAPK) kinase-1/mitogen-activated protein kinase (MAPK) kinase-4, and p38/SAPK-2, phosphorylation occurs between 15 and 30 min after macrophage stimulation with tGPI-mucin or GPI anchors highly purified from tGPI-mucins (tGPI). The use of the specific inhibitors of ERK-1/ERK-2 (PD 98059) and p38/SAPK-2 (SB 203580) phosphorylation also indicates the role of MAPKs, with possible involvement of cAMP response element binding protein, in triggering TNF-alpha and IL-12 synthesis by IFN-gamma-primed-macrophages exposed to tGPI or tGPI-mucin. In addition, tGPI-mucin and tGPI were able to induce phosphorylation of I kappa B, and the use of SN50 peptide, an inhibitor of NF-kappa B translocation, resulted in 70% of TNF-alpha synthesis by macrophages exposed to tGPI-mucin. Finally, the similarity of patterns of MAPK and I kappa B phosphorylation, the concentration of drugs required to inhibit cytokine synthesis, as well as cross-tolerization exhibited by macrophages exposed to tGPI, tGPI-mucin, or bacterial LPS, suggest that receptors with the same functional properties are triggered by these different microbial glycoconjugates.
Assuntos
Citocinas/biossíntese , Glicosilfosfatidilinositóis/metabolismo , Proteínas I-kappa B/metabolismo , Lipopolissacarídeos/farmacologia , MAP Quinase Quinase 4 , Macrófagos/enzimologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Receptores de Superfície Celular/fisiologia , Trypanosoma cruzi/imunologia , Fator 2 Ativador da Transcrição , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/imunologia , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Imidazóis/farmacologia , Tolerância Imunológica/efeitos dos fármacos , Inflamação/imunologia , Interleucina-12/biossíntese , Lipopolissacarídeos/imunologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 8 Ativada por Mitógeno , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Mucinas/imunologia , NF-kappa B/fisiologia , Óxido Nítrico/biossíntese , Fosforilação/efeitos dos fármacos , Proteínas de Protozoários/imunologia , Piridinas/farmacologia , Fatores de Transcrição/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Glycosylphosphatidylinositol (GPI) anchor and glycoinositolphospholipid (GIPL) are abundant molecules present in the membrane of parasitic protozoa that are common etiologic agents of medical and veterinary diseases. Recent studies have documented the immunostimulatory/regulatory activity of protozoan-derived GPI-anchors and related structures. Among the bioactivity displayed by the protozoan-derived GPI-anchor is the ability to elicit the synthesis of pro-inflammatory cytokines as well as nitric oxide by host macrophages. In contrast, at high concentrations GIPL and lipophosphoglycan (LPG) derived from protozoan parasites suppress several functions of the host immune system. Additionally, the protozoan-derived GPI-anchor and GIPL have been shown to serve as targets for both specific B and NK-T lymphocyte responses. This information extends our knowledge about parasite molecules that stimulate/regulate the host immune system during protozoan infection. The identification of receptor(s) and signaling pathways triggered by these GPI-related glycolipids may provide new insights for the development of therapies that inhibit detrimental immune responses or potentiate beneficial immune responses observed during infection with protozoan parasites.
Assuntos
Eucariotos/imunologia , Glicolipídeos/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Fosfolipídeos/metabolismo , Infecções por Protozoários/imunologia , Transdução de Sinais , Animais , Eucariotos/química , Eucariotos/metabolismo , Eucariotos/patogenicidade , Glicolipídeos/química , Glicosilfosfatidilinositóis/química , Humanos , Ativação de Macrófagos , Macrófagos/imunologia , Macrófagos/metabolismo , Fosfolipídeos/química , Infecções por Protozoários/parasitologia , VirulênciaRESUMO
We investigated the kinetics of parasite replication, leukocyte migration, and cytokine/chemokine mRNA expression in the heart tissue from animals infected with the Colombiana strain of Trypanosoma cruzi. Cardiac tissue parasitism was noticeable at 15 days, peaked around 30 days and was dramatically reduced at 120 days postinfection (p.i.). Kinetic studies showed that the inflammatory infiltrate was dominated by the presence of alphabetaT CD3(+ )CD4(+ )CD8(-), alphabetaT CD3(+ )CD4(-)CD8(+ )lymphocytes and macrophages. The mRNA expression of the monokines IL-1beta and IL-12(p40) was elevated at 15 days p.i. and controlled at later time points. In contrast, TNF-alpha mRNA was expressed throughout the infection. Interestingly, we found that at 15 and 30 days p.i. cytokine expression was dominated by the presence of IFN-gamma mRNA, whereas at 60 days or later time points the balance of type 1 and type 2 cytokines was switched in favor of IL-4 and IL-10 mRNAs. The chemokine mRNAs encoding JE, MIP-1alpha, MIP-1beta, KC, and MIP-2 were all mainly expressed at 15 and/or 30 days p.i. and diminished thereafter. In contrast, the expression of RANTES, MIG and IP-10 mRNAs was augmented at 15 days p.i. and persisted at high levels up to 120 days p.i. Taken together, our results indicate that regulation of IFN-gamma and chemokine expression, associated with decreased tissue parasitism, may be largely responsible for the control of inflammation and immunopathology observed in the cardiac tissue of animals infected with T. cruzi.
Assuntos
Cardiomiopatia Chagásica/imunologia , Quimiocinas/genética , Citocinas/genética , Interferon gama/genética , Trypanosoma cruzi/imunologia , Animais , Células Cultivadas , Cardiomiopatia Chagásica/parasitologia , Cardiomiopatia Chagásica/patologia , Quimiocinas CC/genética , Quimiocinas CXC/genética , Modelos Animais de Doenças , Feminino , Expressão Gênica , Coração/parasitologia , Cinética , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/patologia , Parasitemia , RNA MensageiroRESUMO
We compared in vitro and in vivo induction of IL-12 (p40) and IFN-gamma by mouse cells stimulated with Toxoplasma gondii, Trypanosoma cruzi, and different species of Leishmania. Spleen cells cultured in vitro with T. cruzi or T. gondii, but not with Leishmania, produced IL-12 (p40) and IFN-gamma. Accordingly, IL-12 (p40) was produced by macrophages stimulated in vitro with live T. cruzi or T. gondii or membrane glycoconjugates obtained from trypomastigotes or tachyzoites. No IL-12 production was detected when macrophages were stimulated with live parasites or glycoconjugates from Leishmania, regardless of priming with IFN-gamma. In vivo, only T. cruzi and T. gondii induced the synthesis of IL-12 and IFN-gamma by mouse spleen cells after intraperitoneal injection of parasites. When injected subcutaneously, live Leishmania sp. induced IL-12 (p40) and IFN-gamma production by draining lymph node cells, albeit the levels were slightly lower than those induced by infection with T. gondii or T. cruzi using the same route. Together our results indicate that under different conditions, the intracellular protozoa T. gondii and T. cruzi are more potent stimulators of IL-12 and IFN-gamma synthesis by host immune cells than parasites of the genus Leishmania.
Assuntos
Interferon gama/biossíntese , Interleucina-12/biossíntese , Leishmania/imunologia , Toxoplasma/imunologia , Trypanosoma cruzi/imunologia , Animais , Células Cultivadas , Doença de Chagas/imunologia , Feminino , Interleucina-4/biossíntese , Leishmaniose/imunologia , Linfonodos/citologia , Linfonodos/imunologia , Linfonodos/parasitologia , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Baço/citologia , Baço/imunologia , Baço/parasitologia , Toxoplasmose/imunologiaRESUMO
Many persons infected with Toxoplasma gondii develop ocular lesions. Immunologic parameters in the response to T. gondii were evaluated in infected persons with and without ocular lesions and in noninfected controls. Subjects were divided into groups on the basis of presence of serum antibodies to T. gondii, presence of ocular lesions, and clinical history. Production of interleukin-2 and interferon-gamma by peripheral blood mononuclear cells from patients with probable congenital toxoplasmosis was decreased, compared with that in persons with presumed acquired infection. Cell proliferation and delayed-type skin reaction induced by soluble toxoplasma tachyzoite antigen followed the same pattern. Asymptomatic persons showed high levels of interleukin-12 and interferon-gamma, whereas persons with ocular lesions had high interleukin-1 and tumor necrosis factor-alpha responses toward soluble toxoplasma tachyzoite antigen. These data suggest that patients with ocular disease due to congenital infection show tolerance toward the parasite. Furthermore, susceptibility to ocular lesions after acquired toxoplasmosis is associated with high levels of interleukin-1 and tumor necrosis factor-alpha, whereas resistance is associated with high levels of interleukin-12 and interferon-gamma.