RESUMO
Peribunyaviridae is a large family of RNA viruses with several members that cause mild to severe diseases in humans and livestock. Despite their importance in public heath very little is known about the host cell factors hijacked by these viruses to support assembly and cell egress. Here we show that assembly of Oropouche virus, a member of the genus Orthobunyavirus that causes a frequent arboviral infection in South America countries, involves budding of virus particles toward the lumen of Golgi cisternae. As viral replication progresses, these Golgi subcompartments become enlarged and physically separated from Golgi stacks, forming Oropouche viral factory (Vfs) units. At the ultrastructural level, these virally modified Golgi cisternae acquire an MVB appearance, and while they lack typical early and late endosome markers, they become enriched in endosomal complex required for transport (ESCRT) proteins that are involved in MVB biogenesis. Further microscopy and viral replication analysis showed that functional ESCRT machinery is required for efficient Vf morphogenesis and production of infectious OROV particles. Taken together, our results indicate that OROV attracts ESCRT machinery components to Golgi cisternae to mediate membrane remodeling events required for viral assembly and budding at these compartments. This represents an unprecedented mechanism of how viruses hijack host cell components for coordinated morphogenesis.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Orthobunyavirus/metabolismo , Orthobunyavirus/fisiologia , Técnicas de Cultura de Células , Complexos Endossomais de Distribuição Requeridos para Transporte/fisiologia , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Complexo de Golgi/virologia , Células HeLa , Humanos , Orthobunyavirus/crescimento & desenvolvimento , Orthobunyavirus/patogenicidade , Vírion/metabolismo , Montagem de Vírus/fisiologia , Liberação de Vírus/fisiologia , Replicação Viral/fisiologiaRESUMO
Quantitative alterations in mast cell numbers in pancreatic lymph nodes (PLNs) have been reported to be associated with type 1 diabetes (T1D) progression, but their potential role during T1D remains unclear. In this study, we evaluated the role of mast cells in T1D induced by multiple low-dose streptozotocin (MLD-STZ) treatments, using two strains of mast cell-deficient mice (W/W(v) or Wsh/Wsh) and the adoptive transfer of mast cells. Mast cell deficient mice developed severe insulitis and accelerated hyperglycemia, with 100% of mice becoming diabetic compared to their littermates. In parallel, these diabetic mice had decreased numbers of T regulatory (Treg) cells in the PLNs. Additionally, mast cell deficiency caused a significant reduction in IL-10, TGF-ß, and IL-6 expression in the pancreatic tissue. Interestingly, IL-6-deficient mice are more susceptible to T1D associated with reduced Treg-cell numbers in the PLNs, but mast cell transfer from wild-type mice induced protection to T1D in these mice. Finally, mast cell adoptive transfer prior to MLD-STZ administration conferred resistance to T1D, promoted increased Treg cells, and decreased IL-17-producing T cells in the PLNs. Taken together, our results indicate that mast cells are implicated in resistance to STZ-induced T1D via an immunological tolerance mechanism mediated by Treg cells.
Assuntos
Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Tipo 1/imunologia , Regulação da Expressão Gênica/imunologia , Mastócitos/imunologia , Linfócitos T Reguladores/imunologia , Animais , Citocinas/genética , Citocinas/imunologia , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/patologia , Mastócitos/patologia , Camundongos , Camundongos Knockout , Linfócitos T Reguladores/patologia , Células Th17/imunologia , Células Th17/patologiaRESUMO
Snake venom metalloproteinases have been described as responsible for several inflammatory effects. In this study, we investigated the edema and hyperalgesia induced in rats by Batroxase, a P-I metalloproteinase from Bothrops atrox venom, along with possible inflammatory mediators involved in these responses. Batroxase or sterile saline was injected into rat paws and the edema and hyperalgesic effects were evaluated for 6h by using a plethysmometer and a Von Frey system, respectively. Batroxase induced significant edematogenic and hyperalgesic peak responses in the first hours after administration. The inflammatory mediators involved in these responses were assayed by pretreatment of animals with synthesis inhibitors or receptor antagonists. Peak responses were significantly reduced by administration of the glucocorticoid dexamethasone, the H1 receptor antagonist diphenhydramine and the FLAP inhibitor MK-886. Rat paws injected with compound 48/80, a mast cell degranulating agent, followed by Batroxase injection resulted in significant reduction of the edema and hyperalgesia. However, Batroxase itself induced minor degranulation of RBL-2H3 mast cells in vitro. Additionally, the inflammatory responses did not seem to be related to prostaglandins, bradykinin or nitric oxide. Our results indicate a major involvement of histamine and leukotrienes in the edema and hyperalgesia induced by Batroxase, which could be related, at least in part, to mast cell degranulation.
Assuntos
Venenos de Crotalídeos/enzimologia , Edema/patologia , Pé/patologia , Hiperalgesia/patologia , Mediadores da Inflamação/metabolismo , Metaloproteases , Animais , Bothrops , Degranulação Celular/efeitos dos fármacos , Edema/induzido quimicamente , Hiperalgesia/induzido quimicamente , Masculino , Mastócitos/efeitos dos fármacos , Medição da Dor/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
Nef is an HIV-1 accessory protein that promotes viral replication and pathogenesis. A key function of Nef is to ensure sustained depletion of CD4 and MHC-I molecules in infected cells by inducing targeting of these proteins to multivesicular bodies (MVBs), and ultimately to lysosomes for degradation. Nef also affects cellular secretory routes promoting its own secretion via exosomes. To better understand the effects of Nef on the exocytic pathway, we investigated whether this viral factor modifies the composition of exosomes released by T lymphocytes. We showed that both CD4 and MHC-I molecules are secreted in exosomes from T cells and that the expression of Nef reduces the amount of these proteins in exosomes. To investigate the functional role for this novel activity of Nef, we performed in vitro HIV-1 infection assays in the presence of distinct populations of exosomes. We demonstrated that exosomes released by CD4+ T cells, but not CD4- T cells, efficiently inhibit HIV-1 infection in vitro. Because CD4 is the main receptor for HIV-1 infection, these results suggest that CD4 molecules displayed on the surface of exosomes can bind to envelope proteins of HIV-1 hindering virus interaction with target cells and infection. Importantly, CD4-depleted exosomes released by CD4+ T cells expressing Nef have a reduced capacity to inhibit HIV-1 infection in vitro. These results provide evidence that Nef promotes HIV-1 infection by reducing the expression of CD4 in exosomes from infected cells, besides the original role of Nef in reducing the CD4 levels at the cell surface.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Exossomos/imunologia , Produtos do Gene nef/imunologia , Infecções por HIV/imunologia , Linhagem Celular , Regulação para Baixo , Células HEK293 , HIV-1 , Humanos , Complexo Principal de Histocompatibilidade/imunologia , Microscopia de FluorescênciaRESUMO
Previous studies have shown that there is a relationship between periodontal disease and the distribution of collagen fibers. This study evaluated the distribution of collagen types I and III in regenerated bone and periodontal ligament, comparing them to the tissues near the regenerated area and to the healthy periodontium. In the third (P3) and fourth (P4) mandibular premolars of 5 healthy mongrel dogs, bilaterally, buccal class 2 furcation lesions were surgically created and chronified for 3 weeks. After that, full flaps were elevated and expanded polytetrafluoroethylene (e-PTFE) membranes were adapted, sutured and recovered by the flaps. Two weeks after surgery, two membranes on the same side were removed and the other membranes were removed four weeks after surgery. The dogs were euthanized at 12 weeks following placement of the e-PTFE membranes. P3 and P4 teeth as well as the second premolars (healthy control teeth) and their periodontal tissues were removed and histologically processed for Collagen Quantification (COLQ). The amount of type III collagen was higher in native bone compared to the regenerated area. For periodontal ligament, COLQ for type I collagen showed statistically significant differences (Tukeys's Multiple Comparison, p⟨0.05) between the regenerated groups and the control group. These differences were not found for type III COLQ. There are significant differences in collagen distribution among the regenerated, native and control tissues. Membrane removal 2 or 4 weeks postoperatively did not influence the collagen composition.
Assuntos
Osso e Ossos/fisiologia , Colágeno Tipo III/química , Colágeno Tipo I/química , Periodonto/fisiologia , Regeneração , Animais , Materiais Biocompatíveis/química , Regeneração Óssea , Corantes/química , Tecido Conjuntivo/patologia , Cães , Feminino , Masculino , Microscopia , Dente Molar/fisiologia , Ligamento Periodontal/patologia , Ligamento Periodontal/fisiologia , Politetrafluoretileno/química , Fatores SexuaisRESUMO
Eukaryotic translation initiation factor 5A (eIF5A) has a unique character: the presence of an unusual amino acid, hypusine, which is formed by post-translational modifications. Even before the identification of hypusination in eIF5A, the correlation between hypusine formation and protein synthesis, shifting cell proliferation rates, had already been observed. Embryogenesis is a complex process in which cellular proliferation and differentiation are intense. In spite of the fact that many studies have described possible functions for eIF5A, its precise role is under investigation, and to date nothing has been reported about its participation in embryonic development. In this study we show that eIF5A is expressed at all mouse embryonic post-implantation stages with increase in eIF5A mRNA and protein expression levels between embryonic days E10.5 and E13.5. Immunohistochemistry revealed the ubiquitous presence of eIF5A in embryonic tissues and organs at E13.5 day. Interestingly, stronger immunoreactivity to eIF5A was observed in the stomodeum, liver, ectoderm, heart, and eye, and the central nervous system; regions which are known to undergo active differentiation at this stage, suggesting a role of eIF5A in differentiation events. Expression analyses of MyoD, a myogenic transcription factor, revealed a significantly higher expression from day E12.5 on, both at the mRNA and the protein levels suggesting a possible correlation to eIF5A. Accordingly, we next evidenced that inhibiting eIF5A hypusination in mouse myoblast C2C12 cells impairs their differentiation into myotubes and decreases MyoD transcript levels. Those results point to a new functional role for eIF5A, relating it to embryogenesis, development, and cell differentiation.
Assuntos
Desenvolvimento Embrionário/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Camundongos , Fibras Musculares Esqueléticas/citologia , Proteína MyoD/genética , Proteína MyoD/metabolismo , Mioblastos/citologia , Fatores de Iniciação de Peptídeos/antagonistas & inibidores , Fatores de Iniciação de Peptídeos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
Mast Cells (MCs) express toll-like receptor 2 (TLR2), a receptor known to be triggered by several major mycobacterial ligands and involved in resistance against Mycobacterium tuberculosis (MTB) infection. This study investigated whether adoptive transfer of TLR2 positive MCs (TLR2(+/+)) corrects the increased susceptibility of TLR2(-/-) mice to MTB infection. TLR2(-/-) mice displayed increased mycobacterial burden, diminished myeloid cell recruitment and proinflammatory cytokine production accompanied by defective granuloma formation. The reconstitution of these mice with TLR2(+/+) MCs, but not TLR2(-/-), confers better control of the infection, promotes the normalization of myeloid cell recruitment associated with reestablishment of the granuloma formation. In addition, adoptive transfer of TLR2(+/+) MC to TLR2(-/-) mice resulted in regulation of the pulmonary levels of IL-beta, IL-6, TNF-alpha, enhanced Th1 response and activated CD8(+) T cell homing to the lungs. Our results suggest that activation of MCs via TLR2 is required to compensate the defect in protective immunity and inability of TLR2(-/-) mice to control MTB infection.
Assuntos
Mastócitos/imunologia , Mycobacterium tuberculosis , Receptor 2 Toll-Like/metabolismo , Tuberculose Pulmonar/imunologia , Transferência Adotiva , Animais , Linfócitos T CD8-Positivos/imunologia , Movimento Celular , Células Cultivadas , Citocinas/metabolismo , Granuloma/imunologia , Granuloma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor 2 Toll-Like/genética , Tuberculose Pulmonar/prevenção & controleRESUMO
Galectin-3 is a beta-galactoside-binding lectin implicated in the fine-tuning of innate immunity. Rhodococcus equi, a facultative intracellular bacterium of macrophages, causes severe granulomatous bronchopneumonia in young horses and immunocompromised humans. The aim of this study is to investigate the role of galectin-3 in the innate resistance mechanism against R. equi infection. The bacterial challenge of galectin-3-deficient mice (gal3-/-) and their wild-type counterpart (gal3+/+) revealed that the LD50 for the gal3(-/-) mice was about seven times higher than that for the gal3+/+ mice. When challenged with a sublethal dose, gal3(-/-) mice showed lower bacteria counts and higher production of IL-12 and IFN-gamma production, besides exhibiting a delayed although increased inflammatory reaction. Gal3(-/-) macrophages exhibited a decreased frequency of bacterial replication and survival, and higher transcript levels of IL-1beta, IL-6, IL-10, TLR2 and MyD88. R. equi-infected gal3+/+ macrophages showed decreased expression of TLR2, whereas R. equi-infected gal3(-/-) macrophages showed enhanced expression of this receptor. Furthermore, galectin-3 deficiency in macrophages may be responsible for the higher IL-1beta serum levels detected in infected gal3(-/-) mice. Therefore galectin-3 may exert a regulatory role in innate immunity by diminishing IL-1beta production and thus affecting resistance to R. equi infection.
Assuntos
Infecções por Actinomycetales/imunologia , Citocinas/imunologia , Galectina 3/metabolismo , Imunidade Inata , Interleucina-1beta/imunologia , Macrófagos/microbiologia , Infecções por Actinomycetales/microbiologia , Animais , Citocinas/biossíntese , Citocinas/genética , Galectina 3/deficiência , Galectina 3/imunologia , Técnicas de Silenciamento de Genes , Interleucina-12/biossíntese , Interleucina-1beta/biossíntese , Fígado/citologia , Fígado/imunologia , Fígado/microbiologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Rhodococcus equi/imunologia , Baço/citologia , Baço/imunologia , Baço/microbiologia , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/imunologia , Receptor 2 Toll-Like/metabolismoRESUMO
Exposure of endothelial cells to heparin and other antithrombotic drugs specifically stimulates the synthesis of an antithrombotic heparan sulfate (HS). In the present work, biotinylated heparin (BiotHep) was used to characterize the binding site(s) of heparin responsible for the stimulus in HS synthesis on endothelial cells. No differences were observed between biotinylated and non-biotinylated heparin in their ability to increase the synthesis of HS. In kinetic studies the BiotHep showed fast, saturable and specific binding with an apparent K(D) of 83 nM to adherent cells and 44 nM to the extracellular matrix (ECM) in the absence of cells. By confocal and electron microscopy, BiotHep bound only to the ECM, co-localizing with fibronectin. The same pattern of binding to the ECM was observed using heparin conjugated with FITC or Alexa Fluor 488 in the presence or absence of fetal calf serum. However, after degradation of HS, heparin binds to the cell surface, indicating that endogenous HS possibly occupied the heparin binding sites. Analyses by flow cytometry and confocal microscopy of cells with non-associated ECM, showed labeling of the cell surface using syndecan-4 monoclonal antibody as well as wheat germ agglutinin, but no binding of heparin. Furthermore, the stimulation in HS synthesis is not elicited by heparin in the absence of ECM. These results indicate that the stimulus for the synthesis of HS does not require binding of the heparin to the cell surface, and the signaling may be mediated through the ECM.
Assuntos
Células Endoteliais/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Fibrinolíticos/farmacologia , Heparina/análogos & derivados , Heparina/farmacologia , Proteoglicanas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Sítios de Ligação , Biotinilação , Células COS , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Chlorocebus aethiops , Células Endoteliais/metabolismo , Células Endoteliais/ultraestrutura , Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestrutura , Fibrinolíticos/metabolismo , Citometria de Fluxo , Fluoresceína-5-Isotiocianato , Corantes Fluorescentes , Heparina/metabolismo , Humanos , Hidrazinas , Cinética , Ligantes , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Ligação Proteica , Coelhos , Regulação para CimaRESUMO
In vitro, heparin and antithrombotic drugs specifically stimulate the synthesis of an antithrombotic heparan sulfate proteoglycan (HSPG) produced by endothelial cells. The putative heparin binding site(s) that may be related to this phenomenon were investigated. In the preceding article, using various heparin probes, it was shown that the heparin does not bind to the endothelial cell surface, but only to the extracellular matrix. The present study demonstrated that, when the cells were exposed to heparin at 37 degrees C, the heparin was internalized and with time was localized in lysosomes. However, endocytosis of heparin was not required for the stimulation of HSPG synthesis. The requirement for heparin degradation in the stimulus of HSPG synthesis was also investigated. When the cells were incubated with chloroquine, a lysosomotropic amine that raises the lysosomal pH thus inhibiting enzymatic degradation of internalized compounds, stimulation of HSPG synthesis was still observed. These combined results indicate that neither internalization nor degradation of heparin is required for stimulation of HSPG synthesis, and suggests that its binding to the extracellular matrix could be responsible for this effect.