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Production of nitric oxide (NO) by LPS-activated macrophages is due to a complex cellular signaling initiated by TLR4 that leads to the transcription of IFN-ß, which activates IRF-1 and STAT-1, as well as to the activation of NF-κB, required for iNOS transcription. High concentrations of LPS can also be uptaken by scavenger receptors (SRs), which, in concert with TLR4, leads to inflammatory responses. The mechanisms by which TLR4 and SRs interact, and the pathways activated by this interaction in macrophages are not elucidated. Therefore, our main goal was to evaluate the role of SRs, particularly SR-A, in LPS-stimulated macrophages for NO production. We first showed that, surprisingly, LPS can induce the expression of iNOS and the production of NO in TLR4-/- mice, provided exogenous IFN-ß is supplied. These results indicate that LPS stimulate receptors other than TLR4. The inhibition of SR-A using DSS or neutralizing antibody to SR-AI showed that SR-A is essential for the expression of iNOS and NO production in stimulation of TLR4 by LPS. The restoration of the ability to express iNOS and produce NO by addition of rIFN-ß to inhibited SR-A cells indicated that the role of SR-AI in LPS-induced NO production is to provide IFN-ß, probably by mediating the internalization of LPS/TLR4, and the differential inhibition by DSS and neutralizing antibody to SR-AI suggested that other SRs are also involved. Our results reinforce that TLR4 and SR-A act in concert in LPS activation and demonstrated that, for the production of NO, it does mainly by synthesizing IRF-3 and also by activating the TRIF/IRF-3 pathway for IFN-ß production, essential for LPS-mediated transcription of iNOS. Consequently STAT-1 is activated, and IRF-1 is expressed, which together with NF-κB from TLR4/MyD88/TIRAP, induce iNOS synthesis and NO production. SUMMARY SENTENCE: TLR4 and SRs act in concert activating IRF-3 to transcribe IFN-ß and activate STAT-1 to produce NO by LPS-activated macrophages.
Assuntos
NF-kappa B , Óxido Nítrico , Camundongos , Animais , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Receptor 4 Toll-Like/metabolismo , Lipopolissacarídeos , Macrófagos/metabolismo , Receptores Depuradores/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismoRESUMO
Intracellular parasites from the genera Toxoplasma, Plasmodium, Trypanosoma, Leishmania and from the phylum Microsporidia are, respectively, the causative agents of toxoplasmosis, malaria, Chagas disease, leishmaniasis and microsporidiosis, illnesses that kill millions of people around the globe. Crossing the host cell plasma membrane (PM) is an obstacle these parasites must overcome to establish themselves intracellularly and so cause diseases. The mechanisms of cell invasion are quite diverse and include (1) formation of moving junctions that drive parasites into host cells, as for the protozoans Toxoplasma gondii and Plasmodium spp., (2) subversion of endocytic pathways used by the host cell to repair PM, as for Trypanosoma cruzi and Leishmania, (3) induction of phagocytosis as for Leishmania or (4) endocytosis of parasites induced by specialized structures, such as the polar tubes present in microsporidian species. Understanding the early steps of cell entry is essential for the development of vaccines and drugs for the prevention or treatment of these diseases, and thus enormous research efforts have been made to unveil their underlying biological mechanisms. This Review will focus on these mechanisms and the factors involved, with an emphasis on the recent insights into the cell biology of invasion by these pathogens.
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Doença de Chagas , Leishmaniose , Parasitos , Plasmodium , Toxoplasma , Trypanosoma cruzi , AnimaisRESUMO
Intracellular parasites of the genus Leishmania are the causative agents of leishmaniasis. The disease is transmitted by the bite of a sand fly vector, which inoculates the parasite into the skin of mammalian hosts, including humans. During chronic infection the parasite lives and replicates inside phagocytic cells, notably the macrophages. An interesting, but overlooked finding, is that other cell types and even non-phagocytic cells have been found to be infected by Leishmania spp. Nevertheless, the mechanisms by which Leishmania invades such cells had not been previously studied. Here, we show that L. amazonensis can induce their own entry into fibroblasts independently of actin cytoskeleton activity, and, thus, through a mechanism that is distinct from phagocytosis. Invasion involves subversion of host cell functions, such as Ca2+ signaling and recruitment and exocytosis of host cell lysosomes involved in plasma membrane repair.This article has an associated First Person interview with the first author of the paper.
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Membrana Celular/parasitologia , Fibroblastos/parasitologia , Leishmania mexicana , Lisossomos/parasitologia , Citoesqueleto de Actina/parasitologia , Animais , Sinalização do Cálcio , Linhagem Celular , Membrana Celular/metabolismo , Exocitose , Interações Hospedeiro-Parasita , Leishmania mexicana/metabolismo , Leishmania mexicana/parasitologia , Macrófagos/parasitologia , Camundongos , FagocitoseRESUMO
Leishmania (Viannia) guyanensis is one species that causes cutaneous leishmaniasis in the New World. The incidence of infections with this parasite is probably underestimated and few studies exist on this species, despite its epidemiological importance. In particular, there are no studies concerning L. guyanensis metacyclogenesis and no technique for obtaining metacyclic promastigotes for this species is presently available. Here, we have studied L. guyanensis metacyclogenesis in axenic culture, describing the main changes that occur during this process, namely, in morphology and size, sensitivity to complement-mediated lysis, surface carbohydrates and infectivity to macrophages. We have shown that metacyclogenesis in L. guyanensis promastigotes is basically complete on the 4th day of culture, as determined by decreased body size, increased flagellum length, resistance to complement-mediated lysis and infectivity. We have also found that only a fraction of the parasites is agglutinated by Bauhinia purpurea lectin. The non-agglutinated parasites, which also peaked on the 4th day of culture, had all morphological traits typical of the metacyclic stage. This is the first report describing metacyclogenesis in L. guyanensis axenic promastigotes and a simple and efficient method for the purification of metacyclic forms. Furthermore, a model of human macrophage infection with L. guyanensis was established.
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We have previously shown that various species of Leishmania produce a lytic activity, which, in Leishmania amazonensis, is mediated by a pore-forming cytolysin, called leishporin. It is toxic for macrophages in vitro and optimally active at pH 5.0 to 5.5 and at 37 °C, suggesting that it might be active inside phagolysosomes. Leishporin from both L. amazonensis (a-leishporin) and Leishmania guyanensis (g-leishporin) can be activated by proteases, suggesting either a limited proteolysis of an inactive precursor or a proteolytic degradation of a non-covalently linked inhibitor. Here, we show that both a- and g-leishporin are also activated in dissociating conditions, indicating the second hypothesis as the correct one. In fact, we further demonstrated that inactive leishporin is non-covalently associated with an inhibitor, possibly more than one oligopeptide that, when removed, renders leishporin hemolytically active. This activation was shown to be the result of both the improvement of leishporin's ability to bind to phospholipids and the emergence of its pore-forming ability. In vitro results demonstrate that leishporin can be released by the parasites, as they evolve in axenic cultures, in an inactive form that can be activated. These results are compatible with our hypothesis that leishporin can be activated in the protease-rich, low pH, and dissociating environment of parasitophorous vacuoles, leading to disruption of both vacuoles and plasma membranes with the release of amastigotes.
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Eritrócitos/parasitologia , Leishmania/metabolismo , Leishmaniose/parasitologia , Lipídeos de Membrana/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Membrana Celular/metabolismo , Citotoxinas/metabolismo , Ativação Enzimática , Eritrócitos/fisiologia , Hemólise , Humanos , Concentração de Íons de Hidrogênio , Peptídeo Hidrolases/metabolismo , Ligação Proteica , TemperaturaRESUMO
Parasitic protozoa are eukaryotic unicellular organisms that depend on a variety of living organisms and can develop intra- and extracellularly inside their hosts. In humans, these parasites cause diseases with a significant impact on public health, such as malaria, toxoplasmosis, Chagas disease, leishmaniasis and amebiasis. The ability of a parasite in establishing a successful infection depends on a series of intricate evolutionarily selected adaptations, which include the development of molecular and cellular strategies to evade the host immune system effector mechanisms. The complement system is one of the main effector mechanisms and the first humoral shield of hosts innate immunity against pathogens. For unicellular pathogens, such as protozoa, bacteria and fungi, the activation of the complement system may culminate in the elimination of the invader mainly via 1- the formation of a pore that depolarizes the plasma membrane of the parasite, causing cell lysis; 2- opsonization and killing by phagocytes; 3- increasing vascular permeability while also recruiting neutrophils to the site of activation. Numerous strategies to avoid complement activation have been reported for parasitic protozoa, such as 1- sequestration of complement system regulatory proteins produced by the host, 2- expression of complement system regulatory proteins, 3- proteolytic cleavage of different complement effector molecules, 4- formation of a physical glycolipid barrier that prevents deposition of complement molecules on the plasma membrane, and 5- removal, by endocytosis, of complement molecules bound to plasma membrane. In this review, we revisit the different strategies of blocking various stages of complement activation described for the main species of parasitic protozoa, present the most recent discoveries in the field and discuss new perspectives on yet neglected strategies and possible new evasion mechanisms.
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Leishmaniose , Parasitos , Animais , Ativação do Complemento , Proteínas do Sistema Complemento , Homicídio , HumanosRESUMO
Trichoderma spp. are usually considered safe and normally used as biocontrol and biofertilization. Safety for human health is evaluated by several tests that detect various effects such as allergenicity, toxicity, infectivity, and pathogenicity. However, they do not evaluate the effects of the agent upon the immune system. The aim of this study was to investigate the interaction between T. stromaticum spores and mammalian cells to assess the immunomodulatory potential of the spores of this fungus. First, mouse macrophage cell line J774 and human macrophages were exposed to fungal spores and analyzed for structural features, through scanning and transmission electron microscopy. Then, various analysis were performed in human macrophages as to their effect in some functional and molecular aspects of the immune system through immunocytochemistry, flow cytometry and gene expression assays. We demonstrated that T. stromaticum spores induces autophagy and autophagy-related genes (ATGs) and downmodulate inflammatory mediators, including ROS, NLRP3, the cytokines IL-1ß, IL-18, IL-12 and IL-10, as well as TLR2, TLR4, miR-146b and miR-155, which may lead to an augmented susceptibility to pathogens. Our study shows the extension of damages the biofungicide Tricovab® can cause in the innate immune response. Further studies are necessary to elucidate other innate and adaptive immune responses and, consequently, the safety of this fungus when in contact with humans.
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Cell signalling, cell secretion, and plasma membrane repair are processes that critically rely on intracellular vesicles, important components of the endocytic and secretory pathways. More specifically, the strategic distribution of intracellular vesicles is important for diverse cellular processes. The method presented here is a simple, affordable, and efficient tool to analyze the distribution of intracellular vesicles such as lysosomes, endosomes, Golgi vesicles or secretory granules under different experimental conditions. The method is an accessible way to analyze the density and dispersion of intracellular vesicles by combining immunofluorescence with pixel-based quantification software (e.g., ImageJ/FIJI). This protocol can be used widely within the scientific community because it utilizes ImageJ/FIJI, an open source software that is free. By tracking fluorescent vesicles based on their position relative to cell nuclei we are able to quantify and analyze their distribution throughout the cell.
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Leishmania is an intracellular parasite in vertebrate hosts, including man. During infection, amastigotes replicate inside macrophages and are transmitted to healthy cells, leading to amplification of the infection. Although transfer of amastigotes from infected to healthy cells is a crucial step that may shape the outcome of the infection, it is not fully understood. Here we compare L. amazonensis and L. guyanensis infection in C57BL/6 and BALB/c mice and investigate the fate of macrophages when infected with these species of Leishmania in vitro. As previously shown, infection of mice results in distinct outcomes: L. amazonensis causes a chronic infection in both strains of mice (although milder in C57BL/6), whereas L. guyanensis does not cause them disease. In vitro, infection is persistent in L. amazonensis-infected macrophages whereas L. guyanensis growth is controlled by host cells from both strains of mice. We demonstrate that, in vitro, L. amazonensis induces apoptosis of both C57BL/6 and BALB/c macrophages, characterized by PS exposure, DNA cleavage into nucleosomal size fragments, and consequent hypodiploidy. None of these signs were seen in macrophages infected with L. guyanensis, which seem to die through necrosis, as indicated by increased PI-, but not Annexin V-, positive cells. L. amazonensis-induced macrophage apoptosis was associated to activation of caspases-3, -8 and -9 in both strains of mice. Considering these two species of Leishmania and strains of mice, macrophage apoptosis, induced at the initial moments of infection, correlates with chronic infection, regardless of its severity. We present evidence suggestive that macrophages phagocytize L. amazonensis-infected cells, which has not been verified so far. The ingestion of apoptotic infected macrophages by healthy macrophages could be a way of amastigote spreading, leading to the establishment of infection.
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Leishmania/imunologia , Macrófagos/fisiologia , Macrófagos/parasitologia , Animais , Apoptose , Caspase 8/metabolismo , Caspase 9/metabolismo , Permeabilidade da Membrana Celular , Células Cultivadas , Fragmentação do DNA , Diploide , Modelos Animais de Doenças , Progressão da Doença , Leishmania guyanensis , Leishmaniose/imunologia , Leishmaniose/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BLRESUMO
This study was designed to assess in vitro metacyclogenesis of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis clinical field isolates obtained from patient lesions (L. braziliensis IMG3 and PPS6m; L. amazonensis MAB6). Metacyclogenesis was evaluated by different criteria, namely, promastigote size (morphometric analysis and flow cytometry), surface modifications (loss of lectin or monoclonal antibody (mAb) binding, complement resistance), and infectivity to human macrophages. Growth curves were similar for all parasites evaluated. The various features analyzed were expressed in a high percentage of promastigotes at 6th and 10th days of culture and a low percentage at the 2nd day. However, in most isolates, these features, considered as markers of metacyclogenesis, seemed to develop with different time courses, since the percentages of metacyclic forms detected with each technique were usually different. Parasites from 6th or 10th day and those negatively selected with lectin or mAb similarly infected human macrophages. From all isolates analyzed, L. amazonensis PH8 and MAB6 showed the highest and the lowest levels of susceptibility, respectively, to leishmanicidal activity of IFN-γ/LPS-activated macrophages. Our results showed that by using different techniques to evaluate different aspects of metacyclogenesis (morphological and biochemical modifications) different percentages of metacyclic promastigotes can be detected in each isolate culture.
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Proteínas do Sistema Complemento/imunologia , Leishmania braziliensis/citologia , Leishmania braziliensis/imunologia , Estágios do Ciclo de Vida/imunologia , Macrófagos/imunologia , Macrófagos/parasitologia , Anticorpos Monoclonais/imunologia , Células Cultivadas , Humanos , Técnicas In Vitro , Interferon gama/imunologia , Lectinas/imunologia , Leishmania braziliensis/isolamento & purificação , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Lipopolissacarídeos/imunologiaRESUMO
Chromobacterium violaceum, a component of tropical soil microbiota, is an opportunistic pathogenic bacterium that can infect humans and other animals. In addition to identifying a large number of genes that demonstrate the vast biotechnological potential of this bacterium, genome sequencing revealed several virulence factors, including different cytolysins, which can be related to its pathogenicity. Here we confirmed these predictions from genomic analyses by identifying, through mass spectrometry, proteins present in the culture supernatant of C. violaceum that may constitute secreted virulence factors. Among them, we identified a secreted collagenase and the product of a gene with sequence similarity to previously characterized bacterial porins.
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Chromobacterium/metabolismo , Meios de Cultura/química , Fatores de Virulência/análise , Animais , Chromobacterium/genética , Humanos , Espectrometria de Massas , Microbiologia do Solo , Clima Tropical , Fatores de Virulência/genéticaRESUMO
Cutaneous leishmaniasis affects millions of people around the world. Several species of Leishmania infect mouse strains, and murine models closely reproduce the cutaneous lesions caused by the parasite in humans. Mouse models have enabled studies on the pathogenesis and effector mechanisms of host resistance to infection. Here, we review the role of nitric oxide (NO), reactive oxygen species (ROS), and peroxynitrite (ONOO(-)) in the control of parasites by macrophages, which are both the host cells and the effector cells. We also discuss the role of neutrophil-derived oxygen and nitrogen reactive species during infection with Leishmania. We emphasize the role of these cells in the outcome of leishmaniasis early after infection, before the adaptive T(h)-cell immune response.
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To lyse cells, some pore-forming proteins need to bind to receptors on their targets. Studying the binding requirements of Leishmania amazonensis leishporin, we have shown that protease-treated erythrocytes are as sensitive to leishporin-mediated lysis as untreated cells, indicating that protein receptors are dispensable. Similarly, carbohydrate receptors do not seem to be needed, since several sugars do not inhibit leishporin-mediated hemolysis. Conversely, dipalmitoylphosphatidylcholine (DPPC), but not cholesterol, completely inhibits leishporin-mediated lysis. DPPC liposomes, with or without cholesterol, are lysed by leishporin and remove its lytic activity. Our results demonstrate that leishporin is a cholesterol-independent cytolysin that binds directly to phospholipids.