RESUMEN
Hepatopathy is frequently observed in patients with severe malaria but its pathogenesis remains unclear. Galectins are evolutionarily conserved glycan-binding proteins with pleiotropic roles in innate and adaptive immune responses, and exhibit pivotal roles during Plasmodium spp. infection. Here, we analyzed the impact of blockage of galectin-receptor interactions by treatment with alpha (α)-lactose on liver immunopathology during the erythrocytic stage of malaria in mice infected with Plasmodium berghei ANKA (PbANKA). Our results found that compared with PbANKA-infected mice (malarial mice), blockage of galectin-receptor interactions led to decreased host survival rate and increased peripheral blood parasitemia; exacerbated liver pathology, increased numbers of CD68+ macrophages and apoptotic cells, and increased parasite burden in the livers on days 5 and 7 post infection (p.i.) as well as increased mRNA expression levels of galectin-9 (Gal-9) and its receptor, the T cell immunoglobulin domain and mucin domain protein 3 (Tim-3), interferon (IFN)α, IFNγ, and the triggering receptor expressed on myeloid cells (TREM)-1 in the livers or spleens of PbANKA-infected mice on day 7 p.i. Observed by transmission electron microscopy, the peritoneal macrophages isolated from malarial mice with α-lactose treatment had more pseudopodia than those from malarial mice. Measured by using quantitative real-time reverse transcription-polymerase chain reaction assay, the mRNA expression levels of Gal-9, IFNα, IFNß, IFNγ, and TREM-1 were increased in the peritoneal macrophages isolated from malarial mice with α-lactose treatment in comparison of those from malarial mice. Furthermore, significant positive correlations existed between the mRNA levels of Gal-9 and Tim-3/IFNγ/TREM-1 in both the livers and the peritoneal macrophages, and between Gal-9 and Tim-3/TREM-1 in the spleens of malarial mice; significant positive correlations existed between the mRNA levels of Gal-9 and IFNγ in the livers and between Gal-9 and IFNα in the peritoneal macrophages from malarial mice treated with α-lactose. Our data suggest a potential role of galectin-receptor interactions in limiting liver inflammatory response and parasite proliferation by down-regulating the expressions of IFNα, IFNγ, and TREM-1 during PbANKA infection.
Asunto(s)
Eritrocitos/parasitología , Galectinas/fisiología , Hígado/patología , Malaria/patología , Parasitemia/patología , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Femenino , Galectinas/antagonistas & inhibidores , Receptor 2 Celular del Virus de la Hepatitis A/antagonistas & inhibidores , Receptor 2 Celular del Virus de la Hepatitis A/genética , Receptor 2 Celular del Virus de la Hepatitis A/metabolismo , Interferón Tipo I/genética , Interferón Tipo I/metabolismo , Lactosa/farmacología , Lactosa/toxicidad , Hígado/parasitología , Pulmón/metabolismo , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/ultraestructura , Malaria/sangre , Ratones , Plasmodium berghei/crecimiento & desarrollo , Seudópodos/ultraestructura , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptores Inmunológicos/biosíntesis , Receptores Inmunológicos/genética , Receptor Activador Expresado en Células Mieloides 1/biosíntesis , Receptor Activador Expresado en Células Mieloides 1/genéticaRESUMEN
Cystic echinococcosis, an endemic zoonosis in Algeria, is caused by the development of the helminth Echinococcus granulosus. Surgery remains the main treatment despite inducing relapse and several adverse reactions. In this context, natural scolicidal agents seem to be promising tools to overcome these reactions. In our study, we evaluated the phytochemical contents, antioxidant activity and scolicidal effect of Atriplex halimus. In this context, the aqueous extract from AH leaves (AHE) was subjected to preliminary phytochemical screening by HPLC. The in vitro antioxidant activity was determined by DPPH test. The cytotoxicity of AHE was evaluated in murine peritoneal macrophages and cell viability was examined by MTT assay. Moreover, different concentrations of AHE (20, 40, 50, 60 and 100 mg/ml) were tested on E. granulosus protoscoleces (PSC) cultures, during different times of incubation (15, 30, 60, 90, 120 and 180 min). The viability was evaluated by eosin exclusion test. The morphological and ultrastructural damages were evaluated by SEM. Our results indicate that total phenolic and flavonoids contents were 37.93 µg of Gallic acid equivalent per mg of extract (GAE/mg E) and 18.86 µg of Quercetin equivalent per mg (QE/mg E) respectively. Furthermore, AHE has an antioxidant activity with an IC50 of 0.95 mg/ml. Interestingly, the extracts did not exhibit any cytotoxic effect against murine peritoneal macrophages. Moreover, our study indicated a significant scolicidal activity time- and dose-dependent. At 60 and 100 mg/ml; and after 120 min of incubation; the mortality rate was 99.36 and 100%, respectively. The parasite's tegument is one of the plant's targets as demonstrated by SEM. Our findings show the benefits of Atriplex halimus extract as a new promising scolicidal tool in hydatid cyst treatment.
Asunto(s)
Atriplex/química , Echinococcus granulosus/efectos de los fármacos , Extractos Vegetales/farmacología , Animales , Antioxidantes/farmacología , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Echinococcus granulosus/crecimiento & desarrollo , Echinococcus granulosus/ultraestructura , Concentración 50 Inhibidora , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/ultraestructura , Ratones , Microscopía Electrónica de Rastreo , Extractos Vegetales/análisis , Hojas de la Planta/químicaRESUMEN
Cadmium is a highly toxic metal threatening human and animal health. N-acetyl-L-cysteine (NAC) was reported to play a positive role in disease treatment and immune regulation. The present study aimed to explore the effect of NAC administration on Cd-induced cytotoxicity and abnormal immune response on chicken peritoneal macrophages. Peritoneal macrophages isolated from Isa Brown male chickens were exposed to CdCl2 (20 or 50 µM) and/or NAC (500 µM) for different time periods. Results showed that Cd caused dose-dependent damage on chicken peritoneal macrophages characterized by morphologic and ultrastructural alterations, increased cell apoptosis, reactive oxygen species accumulation and mitochondrial injury. Cd exposure inhibited phagocytic activity of chicken peritoneal macrophages, and promoted transcriptional status of pro-inflammatory cytokines (IL-1ß, IL-6 and TNF-α) in both unactivated macrophages and cells in response to lipopolysaccharide (LPS) stimuli. Pretreatment with 500 µM NAC did not affect growth of normal chicken peritoneal macrophages, while remarkably inhibiting Cd-caused cell death, oxidative stress, and mitochondrial membrane depolarization. NAC pretreatment significantly prevented intracellular Cd2+ accumulation in the Cd-exposed macrophages. Inhibitory effects of NAC on Cd-induced ROS accumulation and mitochondrial injury on chicken macrophages were confirmed in HD-11 macrophage cell line. In addition, NAC pretreatment promoted the phagocytic activity of Cd-exposed chicken peritoneal macrophages, and significantly inhibited expression of pro-inflammatory factors (IL-1ß, IL-6 and TNF-α) in both Cd-exposed macrophages and Cd-treated cells in response to LPS stimuli. In conclusion, the present study firstly demonstrated the antagonistic effect of NAC against Cd-caused damage and abnormal immune response on chicken peritoneal macrophages. Protective effect of NAC on chicken macrophages was highly related to its suppression on Cd-induced ROS overproduction, pro-inflammatory reaction and intracellular Cd2+ accumulation.
Asunto(s)
Acetilcisteína/farmacología , Apoptosis/efectos de los fármacos , Cadmio/toxicidad , Pollos , Citocinas/metabolismo , Macrófagos Peritoneales/efectos de los fármacos , Animales , Células Cultivadas , Humanos , Inflamación , Lipopolisacáridos/farmacología , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/ultraestructura , Masculino , Mitocondrias/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
We demonstrated the possibility of penetration of magnetite-gold nanoparticles conjugated with prostate-specific membrane antigen into mouse macrophages. It was found that after 3-h incubation with nanoparticles in a concentration of 15 mg/liter at 37oC, they were seen in only 13% macrophages. In about 90% cells, the nanoparticles were detected within the cytoplasm. Under these conditions, membrane damage was revealed in 25% cells. These results should be taken into account in further development and application of nanomaterials for diagnostic and therapeutic purposes in oncology.
Asunto(s)
Antígenos de Superficie/metabolismo , Muerte Celular/efectos de los fármacos , Medios de Contraste/toxicidad , Glutamato Carboxipeptidasa II/metabolismo , Macrófagos Peritoneales/efectos de los fármacos , Nanopartículas de Magnetita/toxicidad , Animales , Antígenos de Superficie/química , Carbocianinas/química , Línea Celular Tumoral , Medios de Contraste/química , Endocitosis/fisiología , Colorantes Fluorescentes/química , Glutamato Carboxipeptidasa II/química , Oro/química , Humanos , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/ultraestructura , Nanopartículas de Magnetita/química , Nanopartículas de Magnetita/ultraestructura , Masculino , Ratones , Cultivo Primario de Células , Próstata/diagnóstico por imagen , Próstata/metabolismo , Próstata/patología , Unión ProteicaRESUMEN
Lectins are carbohydrate-binding proteins that play important roles in the immune system. Under specific conditions, lectins can form amyloids, proteinaceous aggregates rich in cross ß-strand structures. A Ca++-dependent lectin, isolated from Bothrops leucurus snake venom (BLL) has demonstrated relevant biological activities such as antibacterial and antitumor activity. In this work, we aimed to study the interaction of BLL with macrophages. The formation of amyloid structures by BLL in a cell culture medium, the effects of the lectin on macrophage morphology and cytokine production were investigated. BLL amyloid-like fibrils in RMPI medium, pH 7.2, at 37⯰C was confirmed by binding of Congo Red, Thioflavin T and electron microscopy. Neither binding of amyloid markers nor fibrillar structures were found when the lectin was incubated in RPMI plus galactose, the specific BLL-binding carbohydrate. Several phagocytic compartments containing fibrillar structures were observed in BLL-treated macrophages in RPMI medium for 24â¯h; these compartments showed an apple-green birefringence after Congo Red staining and were positive for thioflavin S and anti-amyloid antibody, indicating the presence of amyloid-like fibrils. No fibrillar material and no labeling were observed when the macrophages were treated with BLL plus galactose or cytochalasin B, an inhibitor of phagocytosis. BLL did not affect the viability of the cells. A significant release of proinflammatory (TNF-α, IL-6, INF-Ï and IL-1ß) and regulatory (IL-10) cytokines was observed in BLL-treated macrophages. Taken together, our results shed light on the structural organization of BLL, improving knowledge about the interaction of lectin with macrophages. The phagocytosis of amyloid-like aggregates together with the proinflammatory response induced by BLL may open new perspectives for the use of this lectin as an interesting model to study cytokines and the production of other mediators as well as understand the mechanisms occurring in human immune cells during amyloid protein deposition.
Asunto(s)
Bothrops , Venenos de Crotálidos/farmacología , Lectinas Tipo C/química , Macrófagos Peritoneales/citología , Amiloide/química , Amiloide/metabolismo , Animales , Venenos de Crotálidos/química , Citocalasina B , Citocinas/metabolismo , Galactosa/química , Lectinas Tipo C/metabolismo , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/ultraestructura , Ratones Endogámicos BALB C , FagocitosisRESUMEN
By using near-infrared fluorescent protein (iRFP)-expressing hematopoietic cells, we established a novel, quantitative, in vivo, noninvasive atherosclerosis imaging system. This murine atherosclerosis imaging approach targets macrophages expressing iRFP in plaques. Low-density lipoprotein receptor-deficient (LDLR-/-) mice transplanted with beta-actin promoter-derived iRFP transgenic (TG) mouse bone marrow (BM) cells (iRFP â LDLR-/-) were used. Atherosclerosis was induced by a nonfluorescent 1.25% cholesterol diet (HCD). Atherosclerosis was compared among the three differently induced mouse groups. iRFP â LDLR-/- mice fed a normal diet (ND) and LDLR-/- mice transplanted with wild-type (WT) BM cells were used as controls. The in vivo imaging system (IVIS) detected an enhanced iRFP signal in the thoracic aorta of HCD-fed iRFP â LDLR-/- mice, whereas iRFP signals were not observed in the control mice. Time-course imaging showed a gradual increase in the signal area, which was correlated with atherosclerotic plaque progression. Oil red O (ORO) staining of aortas and histological analysis of plaques confirmed that the detected signal was strictly emitted from plaque-positive areas of the aorta. Our new murine atherosclerosis imaging system can noninvasively image atherosclerotic plaques in the aorta and generate longitudinal data, validating the ability of the system to monitor lesion progression.
Asunto(s)
Enfermedades de la Aorta/diagnóstico por imagen , Aterosclerosis/diagnóstico por imagen , Mediciones Luminiscentes/métodos , Proteínas Luminiscentes/análisis , Imagen Óptica/métodos , Placa Aterosclerótica/diagnóstico por imagen , Actinas/genética , Animales , Enfermedades de la Aorta/genética , Aterosclerosis/etiología , Aterosclerosis/genética , Compuestos Azo , Trasplante de Médula Ósea , Colesterol en la Dieta/toxicidad , Colorantes , Citometría de Flujo , Genes Reporteros , Genes Sintéticos , Proteínas Luminiscentes/genética , Macrófagos Peritoneales/química , Macrófagos Peritoneales/ultraestructura , Ratones , Ratones Transgénicos , Microscopía Fluorescente , Regiones Promotoras Genéticas , Receptores de LDL/deficiencia , Proteínas Recombinantes/análisis , Proteínas Recombinantes/genéticaRESUMEN
Macrophage functions in the immune response depend on their ability to infiltrate tissues and organs. The penetration between and within the tissues requires degradation of extracellular matrix (ECM), a function performed by the specialized, endopeptidase- and actin filament- rich organelles located at the ventral surface of macrophage, called the podosomes. Podosome formation requires local inhibition of small GTPase RhoA activity, and depends on Rac 1/Rho guanine nucleotide exchange factor 7, ß-PIX and its binding partner the p21-activated kinase (PAK-1). The activity of RhoA and Rac 1 is in turn regulated by mTOR/mTORC2 pathway. Here we showed that a fungus metabolite Fingolimod (FTY720, Gilenya), which is clinically approved for the treatment of multiple sclerosis, down-regulates Rictor, which is a signature molecule of mTORC2 and dictates its substrate (actin cytoskeleton) specificity, down-regulates RhoA, up-regulates PAK-1, and causes amplification of podosomes in mouse peritoneal macrophages.
Asunto(s)
Matriz Extracelular/metabolismo , Macrófagos Peritoneales/fisiología , Esclerosis Múltiple/inmunología , Podosomas/metabolismo , Quinasas p21 Activadas/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Células Cultivadas , Femenino , Clorhidrato de Fingolimod/farmacología , Macrófagos Peritoneales/ultraestructura , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Ratones , Ratones Endogámicos C57BL , Podosomas/ultraestructura , Embarazo , Proteína Asociada al mTOR Insensible a la Rapamicina/metabolismoRESUMEN
BACKGROUND: Telocytes (TCs), a recently discovered novel type of interstitial cells, were also found in a wide variety of human and mammalian reproductive organs/tissues, including uterus, oviduct and placenta. Previously, we demonstrated that TCs-conditioned media was capable of activating peritoneal macrophages (pMACs) through paracrine effects. This study investigates the hypothesis that direct interaction of TCs with pMACs will also play a significant role in immunoregulation of pMACs. METHODS: TCs and pMACs were derived from the uterus and intraperitoneal cavity of female BALB/c mice, respectively. TCs were identified by immunofluorescence and then co-cultured directly with pMACs for 24 h without added cytokines, to observe the in vitro biological behavior of pMACs. We used histochemical staining to study morphology and mitochondrial metabolism of pMACs, scanning electron microscopy to study heterocellular junctions, flow cytometry to investigate mitochondrial membrane potential (ΔΨm) and apoptosis, and transwell chambers to study invasion ability. Student-t test was used accordingly. RESULTS: Presently, TCs with typical structure and immunophenotype of double CD-34-positive/vimentin-positive were successfully isolated. pMACs co-cultured with TCs showed obviously morphological activation, with enhanced energy metabolism (P < 0.05). Meanwhile, direct physical cell-to-cell interaction promoted the development of heterocellular junctions between TCs and pMACs. Furthermore, TCs treatment markedly reduced the depletion of ΔΨm in co-cultured pMACs (all P < 0.05), and inhibited their apoptosis (P < 0.05). Functionally, pMACs co-cultured with TCs showed enhanced invasion ability (P < 0.05). CONCLUSIONS: Direct physical cell-to-cell interaction promoted the development of heterocellular junctions between TCs and pMACs, presumably responsible for the observed novel efficient way of pMACs activation via mitochondrial signaling pathway. TCs-educated pMACs might be a promising way to restore the defective immunosurveillance in endometriosis (EMs), led to the enhanced treatment efficacy of EMs in a simple and clinically feasible fashion.
Asunto(s)
Apoptosis , Movimiento Celular , Forma de la Célula , Macrófagos Peritoneales/citología , Telocitos/citología , Animales , Separación Celular , Técnicas de Cocultivo , Metabolismo Energético , Femenino , Macrófagos Peritoneales/ultraestructura , Potencial de la Membrana Mitocondrial , Ratones Endogámicos BALB C , Telocitos/ultraestructuraRESUMEN
Macrophages play crucial role in tissue homeostasis and the innate and adaptive immune response. Depending on the state of activation macrophages acquire distinct phenotypes that depend on actin, which is regulated by small GTPase RhoA. The naive M0 macrophages are slightly elongated, pro-inflammatory M1 are round and M2 anti-inflammatory macrophages are elongated. We showed previously that interference with RhoA pathway (RhoA deletion or RhoA/ROCK kinase inhibition) disrupted actin, produced extremely elongated (hummingbird) macrophage phenotype and inhibited macrophage movement toward transplanted hearts. The RhoA function depends on the family of guanine-nucleotide exchange factors (GEFs), which catalyze the exchange of GDP for GTP and activate RhoA that reorganizes actin cytoskeleton. Using actin staining, immunostaining, Western blotting, flow cytometry and transmission electron microscopy we studied how a direct inhibition of Rho-GEFs with Rhosin (Rho GEF-binding domain blocker) and Y16 (Rho GEF DH-PH domain blocker) affects M0, M1 and M2 macrophage phenotypes. We also studied how Rho-GEFs inhibition and RhoA deletion affects organization of Golgi complex that is crucial for normal macrophage functions such as phagocytosis, antigen presentation and receptor recycling. We found that GEFs inhibition differently affected M0, M1 and M2 macrophages phenotype and that GEFs inhibition and RhoA deletion both caused changes in the ultrastructure of the Golgi complex. These results suggest that actin/RhoA- dependent shaping of macrophage phenotype has different requirements for activity of RhoA/GEFs pathway in M0, M1 and M2 macrophages, and that RhoA and Rho-GEFs functions are necessary for the maintenance of actin-dependent organization of Golgi complex.
Asunto(s)
Aparato de Golgi/metabolismo , Macrófagos Peritoneales/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Animales , Aparato de Golgi/genética , Aparato de Golgi/ultraestructura , Macrófagos Peritoneales/ultraestructura , Ratones , Ratones Transgénicos , Compuestos Orgánicos/farmacología , Fagocitosis , Factores de Intercambio de Guanina Nucleótido Rho/genética , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismo , Quinasas Asociadas a rho/genética , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoARESUMEN
Activated macrophages have a greater ability of phagocytosis against pathogens that is mediated by large-scale actin rearrangement. However, molecular machineries that conduct this task have not been fully identified. Here, we demonstrate an unanticipated role of TAGLN2, a 22-kDa actin-binding protein, in Toll-like receptor (TLR)-stimulated phagocytosis. TAGLN2 was greatly induced in macrophages in response to lipopolysaccharide (LPS), a ligand for TLR4, partly via the NF-κB pathway. TAGLN2-deficient macrophages (TAGLN2 -/-) showed defective phagocytic functions of IgM- and IgG-coated sheep red blood cells as well as bacteria. Cell signaling pathways involved in actin rearrangement-PI3 kinase/AKT and Ras-ERK-were also down-regulated in LPS-stimulated TAGLN2-deficient macrophages. Moreover, TAGLN2 -/- mice showed higher mortality after bacterial infection than wild-type littermates. Thus, our results revealed a novel function of TAGLN2 as a molecular armament required for host defense.
Asunto(s)
Lipopolisacáridos/farmacología , Activación de Macrófagos/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas Musculares/metabolismo , Fagocitosis/efectos de los fármacos , Actinas/metabolismo , Animales , Extensiones de la Superficie Celular/metabolismo , Susceptibilidad a Enfermedades , Humanos , Células Jurkat , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/ultraestructura , Ratones , Ratones Endogámicos C57BL , Proteínas de Microfilamentos/deficiencia , Proteínas Musculares/deficiencia , Peritonitis/microbiología , Peritonitis/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Polimerizacion , Proteínas Proto-Oncogénicas c-akt/metabolismo , Células RAW 264.7 , Análisis de SupervivenciaRESUMEN
Macrophages belong to the innate immune cells and play a key role in the pathogenesis of viral infections. The results of ultrastructural study of macrophages infected with tick-borne encephalitis virus (TBEV), the Flavivirus family, pathogens of human infections, affecting the nervous system, were presented. With the assistance of virological methods was found that the TBEV are absorbed by macrophages and replication in them. An ultrastructural study has shown that the virus enters into the cytoplasm by local destruction of plasmalemma and newly synthesized virus particles exited from the cell by same. Simultaneously there is a seal of perinuclear cytoplasm space, where found in a large number of ribosomes, microfilaments, ribonucleoprotein fibers and viral special structure: nucleocapsids, tubular formations and viral layers (fabrics). On the surface of last structures the newly synthesized virus particles were visualized. Thus, the evidence shows that macrophages play a role in the spread of TBEV, being for their the target cell. As active antigen presenting cells the macrophages can modulate the protective response of the body and influence on the pathogenesis of tick-borne encephalitis.
Asunto(s)
Virus de la Encefalitis Transmitidos por Garrapatas , Encefalitis Transmitida por Garrapatas , Macrófagos Peritoneales , Animales , Virus de la Encefalitis Transmitidos por Garrapatas/metabolismo , Virus de la Encefalitis Transmitidos por Garrapatas/ultraestructura , Encefalitis Transmitida por Garrapatas/metabolismo , Encefalitis Transmitida por Garrapatas/patología , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/ultraestructura , Macrófagos Peritoneales/virología , RatonesRESUMEN
Leishmaniasis are a neglected group of emerging diseases that have been found in 98 countries and are caused by protozoa of the genus Leishmania. The therapy for leishmaniasis causes several side effects and leads to drug-resistant strains. Natural products from plants have exhibited activities against Leishmania in various experimental models. Physalis angulata is a widely used plant in popular medicine, and in the literature it has well-documented leishmanicidal activity. However, its mechanism of action is still unknown. Thus, this study aims to evaluate the mechanism driving the leishmanicidal activity of an aqueous extract of P. angulata root (AEPa). AEPa was effective against both promastigotes and intracellular amastigote forms of Leishmania amazonensis. This effect was mediated by an increase of reactive oxygen species (ROS), but not of nitric oxide (NO). The increased production of ROS induces cell death by phenotypes seems by apoptosis cell death in Leishmania, but not autophagy or necrosis. In addition, morphological analysis of macrophages showed that AEPa induced a high number of cytoplasmic projections, increased the volume of cytoplasm and number of vacuoles, caused cytoskeleton alterations and resulted in high spreading ability. AEPa also promoted superoxide anion (O2(-)) production in both uninfected macrophages and those infected with Leishmania. Therefore, these results revealed that AEPa causes cell death by phenotypes seems by apoptosis cell death in L. amazonensis and modulates macrophage activation through morphofunctional alterations and O2(-) generation to induce Leishmania death.
Asunto(s)
Leishmania/fisiología , Macrófagos Peritoneales/efectos de los fármacos , Physalis/química , Extractos Vegetales/farmacología , Raíces de Plantas/química , Especies Reactivas de Oxígeno/metabolismo , Animales , Apoptosis/efectos de los fármacos , Autofagia , Leishmania/efectos de los fármacos , Leishmania/inmunología , Estadios del Ciclo de Vida/efectos de los fármacos , Activación de Macrófagos/efectos de los fármacos , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/parasitología , Macrófagos Peritoneales/ultraestructura , Ratones , Necrosis/parasitología , FitoterapiaRESUMEN
Macrophage migration to the focus of infection is a hallmark of the innate immune response. Macrophage spreading, adhesion, and migration through the extracellular matrix require dynamic remodeling of the actin cytoskeleton associated to integrin clustering in podosomes and focal adhesions. Here, we show that prostaglandin E2 (PGE2 ), the main prostaglandin produced by macrophages during inflammation, promote the distinctive dose-dependent formation of podosomes or focal adhesions in macrophages. Low concentrations of PGE2 increased p110γ PI3K expression, phosphorylation of actin-related protein 2, and formation of podosomes, which enhanced macrophage migration in response to chemokines. However, high doses of PGE2 increased phosphorylation of paxillin and focal adhesion kinase, the expression of serine/threonine protein kinase 1, and promoted focal adhesion formation and macrophage adhesion, reducing macrophage chemotaxis. In summary, we describe the dual role of PGE2 as a promoter of macrophage chemotaxis and adhesion, proposing a new model of macrophage migration to the inflammatory focus in the presence of a gradient of PGE2 .
Asunto(s)
Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Dinoprostona/farmacología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/fisiología , Proteína 2 Relacionada con la Actina/genética , Proteína 2 Relacionada con la Actina/metabolismo , Animales , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Adhesiones Focales/efectos de los fármacos , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/ultraestructura , Ratones , Paxillin/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Podosomas/efectos de los fármacos , Proteínas Quinasas/genética , Transducción de Señal/efectos de los fármacosRESUMEN
Infection by the protozoan parasite Toxoplasma gondii is highly prevalent worldwide and may have serious clinical manifestations in immunocompromised patients. T. gondii is an obligate intracellular parasite that infects almost any cell type in mammalian hosts, including immune cells. The immune cells express purinergic P2 receptors in their membrane--subdivided into P2Y and P2X subfamilies--whose activation is important for infection control. Here, we examined the effect of treatment with UTP and UDP in mouse peritoneal macrophages infected with T. gondii tachyzoites. Treatment with these nucleotides reduced parasitic load by 90%, but did not increase the levels of the inflammatory mediators NO and ROS, nor did it modulate host cell death by apoptosis or necrosis. On the other hand, UTP and UDP treatments induced early egress of tachyzoites from infected macrophages, in a Ca2+-dependent manner, as shown by scanning electron microscopy analysis, and videomicroscopy. In subsequent infections, prematurely egressed parasites had reduced infectivity, and could neither replicate nor inhibit the fusion of lysosomes to the parasitophorous vacuole. The use of selective agonists and antagonists of the receptor subtypes P2Y2 and P2Y4 and P2Y6 showed that premature parasite egress may be mediated by the activation of these receptor subtypes. Our results suggest that the activity of P2Y host cell receptors controls T. gondii infection in macrophages, highlighting the importance of pyrimidinergic signaling for innate immune system response against infection. Finally the P2Y receptors should be considered as new target for the development of drugs against T. gondii infection.
Asunto(s)
Macrófagos Peritoneales/parasitología , Macrófagos/parasitología , Receptores Purinérgicos P2Y/metabolismo , Toxoplasma/fisiología , Animales , Apoptosis/efectos de los fármacos , Células Cultivadas , Femenino , Interacciones Huésped-Parásitos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/ultraestructura , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/ultraestructura , Masculino , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Óxido Nítrico/metabolismo , Agonistas del Receptor Purinérgico P2Y/farmacología , Antagonistas del Receptor Purinérgico P2Y/farmacología , Especies Reactivas de Oxígeno/metabolismo , Suramina/farmacología , Uridina Difosfato/farmacología , Uridina Trifosfato/farmacologíaRESUMEN
Fullerenes have been applied to various fields in recent years for their unique physicochemical properties. C60's potential environmental and health risks are therefore being investigated. This work presents the cytotoxicity of aqueous C60 aggregates (nC60) by using the mouse peritoneal macrophage (RAW264.7) as a model biological system. Cells incubated with nC60 showed a decrease in cell viability, and the quantities of cell apoptosis increased significantly in a dose-dependent and time-dependent manner. By TEM imaging cells were observed to be asymmetric and shrunken compared to control cells. nC60 was adsorbed onto the plasma membrane and apparent phagocytosis was observed after a 24h exposure. The intracellular reactive oxygen species (ROS) were quantified with the dichlorofluorescein diacetate (DCFH-DA) detection assay kit by flow cytometry and found to increase in treated cells. Membrane damage and lipid peroxidation were not observed, since both intracellular and extracellular MDA showed no variation. Detection of the mitochondrial membrane potential (MMP) by JC-1 assay kit showed that MMP had an obvious dose-dependent loss. We speculate that the nC60 aggregates induced apoptosis of macrophage by changing the mitochondrial membrane potential.
Asunto(s)
Fulerenos/toxicidad , Macrófagos Peritoneales/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , L-Lactato Deshidrogenasa/metabolismo , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/ultraestructura , Ratones , Microscopía Electrónica de Transmisión , Necrosis/inducido químicamente , Especies Reactivas de Oxígeno/metabolismo , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismoRESUMEN
Lipid bodies [lipid droplets (LBs)] are lipid-rich organelles involved in lipid metabolism, signalling and inflammation. Recent findings suggest a role for LBs in host response to infection; however, the potential functions of this organelle in Toxoplasma gondii infection and how it alters macrophage microbicidal capacity during infection are not well understood. Here, we investigated the role of host LBs in T. gondii infection in mouse peritoneal macrophages in vitro. Macrophages cultured with mouse serum (MS) had higher numbers of LBs than those cultured in foetal bovine serum and can function as a model to study the role of LBs during intracellular pathogen infection. LBs were found in association with the parasitophorous vacuole, suggesting that T. gondii may benefit from this lipid source. Moreover, increased numbers of macrophage LBs correlated with high prostaglandin E2 (PGE2) production and decreased nitric oxide (NO) synthesis. Accordingly, LB-enriched macrophages cultured with MS were less efficient at controlling T. gondii growth. Treatment of macrophages cultured with MS with indomethacin, an inhibitor of PGE2 production, increased the microbicidal capacity against T. gondii. Collectively, these results suggest that culture with MS caused a decrease in microbicidal activity of macrophages against T. gondii by increasing PGE2 while lowering NO production.
Asunto(s)
Gotas Lipídicas/parasitología , Activación de Macrófagos/fisiología , Macrófagos Peritoneales/parasitología , Toxoplasma/fisiología , Vacuolas/parasitología , Animales , Bovinos , Interacciones Huésped-Parásitos , Indometacina/farmacología , Gotas Lipídicas/fisiología , Macrófagos Peritoneales/química , Macrófagos Peritoneales/fisiología , Macrófagos Peritoneales/ultraestructura , Masculino , Ratones , Ratones Endogámicos C3H , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Óxido Nítrico/biosíntesis , Cultivo Primario de Células , Prostaglandinas E/antagonistas & inhibidores , Prostaglandinas E/biosíntesis , Vacuolas/fisiologíaRESUMEN
Cellular TG stores are efficiently hydrolyzed by adipose TG lipase (ATGL). Its coactivator comparative gene identification-58 (CGI-58) strongly increases ATGL-mediated TG catabolism in cell culture experiments. To investigate the consequences of CGI-58 deficiency in murine macrophages, we generated mice with a targeted deletion of CGI-58 in myeloid cells (macCGI-58(-/-) mice). CGI-58(-/-) macrophages accumulate intracellular TG-rich lipid droplets and have decreased phagocytic capacity, comparable to ATGL(-/-) macrophages. In contrast to ATGL(-/-) macrophages, however, CGI-58(-/-) macrophages have intact mitochondria and show no indications of mitochondrial apoptosis and endoplasmic reticulum stress, suggesting that TG accumulation per se lacks a significant role in processes leading to mitochondrial dysfunction. Another notable difference is the fact that CGI-58(-/-) macrophages adopt an M1-like phenotype in vitro. Finally, we investigated atherosclerosis susceptibility in macCGI-58/ApoE-double KO (DKO) animals. In response to high-fat/high-cholesterol diet feeding, DKO animals showed comparable plaque formation as observed in ApoE(-/-) mice. In agreement, antisense oligonucleotide-mediated knockdown of CGI-58 in LDL receptor(-/-) mice did not alter atherosclerosis burden in the aortic root. These results suggest that macrophage function and atherosclerosis susceptibility differ fundamentally in these two animal models with disturbed TG catabolism, showing a more severe phenotype by ATGL deficiency.
Asunto(s)
1-Acilglicerol-3-Fosfato O-Aciltransferasa/metabolismo , Aterosclerosis/metabolismo , Eliminación de Gen , Lipasa/metabolismo , Macrófagos Peritoneales/inmunología , Fagocitosis , 1-Acilglicerol-3-Fosfato O-Aciltransferasa/antagonistas & inhibidores , 1-Acilglicerol-3-Fosfato O-Aciltransferasa/genética , Animales , Apoptosis , Aterosclerosis/etiología , Aterosclerosis/inmunología , Aterosclerosis/patología , Células Cultivadas , Cruzamientos Genéticos , Dieta Alta en Grasa/efectos adversos , Femenino , Técnicas de Silenciamiento del Gen , Lipasa/genética , Gotas Lipídicas/inmunología , Gotas Lipídicas/metabolismo , Gotas Lipídicas/ultraestructura , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/ultraestructura , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Mitocondrias/inmunología , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Oligonucleótidos Antisentido/administración & dosificación , Triglicéridos/metabolismoRESUMEN
Lipid bodies [lipid droplets (LBs)] are lipid-rich organelles involved in lipid metabolism, signalling and inflammation. Recent findings suggest a role for LBs in host response to infection; however, the potential functions of this organelle in Toxoplasma gondii infection and how it alters macrophage microbicidal capacity during infection are not well understood. Here, we investigated the role of host LBs in T. gondii infection in mouse peritoneal macrophages in vitro. Macrophages cultured with mouse serum (MS) had higher numbers of LBs than those cultured in foetal bovine serum and can function as a model to study the role of LBs during intracellular pathogen infection. LBs were found in association with the parasitophorous vacuole, suggesting that T. gondii may benefit from this lipid source. Moreover, increased numbers of macrophage LBs correlated with high prostaglandin E2 (PGE2) production and decreased nitric oxide (NO) synthesis. Accordingly, LB-enriched macrophages cultured with MS were less efficient at controlling T. gondii growth. Treatment of macrophages cultured with MS with indomethacin, an inhibitor of PGE2 production, increased the microbicidal capacity against T. gondii. Collectively, these results suggest that culture with MS caused a decrease in microbicidal activity of macrophages against T. gondii by increasing PGE2 while lowering NO production.
Asunto(s)
Animales , Bovinos , Masculino , Ratones , Gotas Lipídicas/parasitología , Activación de Macrófagos/fisiología , Macrófagos Peritoneales/parasitología , Toxoplasma/fisiología , Vacuolas/parasitología , Interacciones Huésped-Parásitos , Indometacina/farmacología , Gotas Lipídicas/fisiología , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Macrófagos Peritoneales/química , Macrófagos Peritoneales/fisiología , Macrófagos Peritoneales/ultraestructura , Óxido Nítrico/biosíntesis , Cultivo Primario de Células , Prostaglandinas E/antagonistas & inhibidores , Prostaglandinas E/biosíntesis , Vacuolas/fisiologíaRESUMEN
In the present study, we assessed morphological changes and cytokine production after in vitro interaction with causative agents of American cutaneous leishmaniasis and compared the microglia and macrophage immune responses. Cultures of microglia and macrophages infected with stationary-phase promastigotes of Leishmania (Viannia) shawi, Leishmania (Viannia) braziliensis or Leishmania (Leishmania) amazonensis were evaluated 24, 48 and 72 h after interaction. Macrophages only presented the classical phagocytic process while microglia also displayed large cytoplasmic projections similar to the ruffles described in macropinocytosis. In the macrophage cultures, the percentage of infected cells increased over time, in a fashion that was dependent on the parasite species. In contrast, in microglial cells as the culture time progressed, there was a significant reduction in the percentage of infected cells independent of parasite species. Measurements of cytokines in macrophage cultures 48 h after interactions revealed distinct expression patterns for different parasites, whereas in microglial cultures they were similar for all Leishmania tested species. Taken together, our results suggest that microglia may have a higher phagocytic ability and cytotoxic potential than macrophages for all investigated species. The robust response of microglia against all parasite species may suggest microglia have an important role in the defence against cerebral leishmaniasis.
Asunto(s)
Citocinas/metabolismo , Leishmania/fisiología , Leishmaniasis/inmunología , Macrófagos Peritoneales/inmunología , Microglía/inmunología , Animales , Supervivencia Celular , Células Cultivadas , Femenino , Leishmaniasis/parasitología , Macrófagos Peritoneales/ultraestructura , Ratones , Ratones Endogámicos BALB C , Microglía/ultraestructura , Óxido Nítrico/metabolismo , FagocitosisRESUMEN
Heat shock protein 70 is highly conserved cytosolic protein which have important role in growth, development, and apoptosis. Hsp70 is well-known activator of macrophages and enhances the release of specific and non-specific effector molecules that have major role in tumor destruction and immunopotentiation of host. However, morphological effects of hsp 70 has not been carried out, therefore, morphological effects of hsp 70 on murine peritoneal macrophages were examined by light microscopy and scanning electron microscopy. Thioglycolate-induced peritoneal macrophages were prepared from BALB/c mice and cultured for 24 h in the presence of the hsp70. Tumor-associated macrophages treated with 10 µg/ml were varied in shape, mostly spindle shaped, i.e., stretched bidirectionally; surface ruffles were increased and their lamellipodia was prominent which suggest that hsp 70 treatment not only enhances the functional state of the peritoneal macrophages but also initiate immense morphological changes leading to increased endothelium adherence, increased antigen uptake, and increased migration to the inflammatory site.