RESUMO
Outer membrane vesicles (OMVs) released by some gram-negative bacteria have been shown to exert immunomodulatory effects that favor the establishment of the infection. The aim of the present study was to assess the interaction of OMVs from Brucella abortus with human epithelial cells (HeLa) and monocytes (THP-1), and the potential immunomodulatory effects they may exert. Using confocal microscopy and flow cytometry, FITC-labeled OMVs were shown to be internalized by both cell types. Internalization was shown to be partially mediated by clathrin-mediated endocytosis. Pretreatment of THP-1 cells with Brucella OMVs inhibited some cytokine responses (TNF-α and IL-8) to E. coli LPS, Pam3Cys or flagellin (TLR4, TLR2 and TLR5 agonists, respectively). Similarly, pretreatment with Brucella OMVs inhibited the cytokine response of THP-1 cells to B. abortus infection. Treatment of THP-1 cells with OMVs during IFN-γ stimulation reduced significantly the inducing effect of this cytokine on MHC-II expression. OMVs induced a dose-dependent increase of ICAM-1 expression on THP-1 cells and an increased adhesion of these cells to human endothelial cells. The addition of OMVs to THP-1 cultures before the incubation with live B. abortus resulted in increased numbers of adhered and internalized bacteria as compared to cells not treated with OMVs. Overall, these results suggest that OMVs from B. abortus exert cellular effects that promote the internalization of these bacteria by human monocytes, but also downregulate the innate immune response of these cells to Brucella infection. These effects may favor the persistence of Brucella within host cells.
Assuntos
Brucella abortus/imunologia , Brucella abortus/metabolismo , Imunidade Inata , Monócitos/imunologia , Fagocitose/imunologia , Vesículas Transportadoras/metabolismo , Aderência Bacteriana/imunologia , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Clatrina/metabolismo , Citocinas/metabolismo , Endocitose/fisiologia , Células HeLa , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Interferon gama/farmacologia , Monócitos/efeitos dos fármacos , Monócitos/microbiologia , Receptores Toll-Like/agonistas , Receptores Toll-Like/metabolismo , Vesículas Transportadoras/imunologiaRESUMO
Microtubule cytoskeleton is a dynamic structure involved in the maintenance of eukaryote cell shape, motion of cilia and flagellum, and intracellular movement of vesicles and organelles. Many antibodies against tubulins have been described, most of them against the C-terminal portion, which is exposed at the outside of the microtubules. By generating a novel set of monoclonal antibodies against the cytoskeleton of Trypanosoma cruzi, a flagellate protozoan that causes Chagas' disease, we selected a clone (mAb 3G4) that recognizes ß-tubulin. The epitope for mAb 3G4 was mapped by pepscan to a highly conserved sequence motif found between α-helices 11 and 12 of the C-terminus of ß-tubulin in eukaryotes. It labels vesicular structures in both T. cruzi and mammalian cells, colocalizing respectively with a major cysteine protease (Cruzipain) and lysosome associated protein (LAMP2) respectively, but it does not label regular microtubules on these cellular models. We propose that the epitope recognized by mAb 3G4 is exposed only in a form of tubulin associated with endosomes.
Assuntos
Anticorpos Monoclonais/metabolismo , Vesículas Transportadoras/metabolismo , Trypanosoma cruzi/metabolismo , Tubulina (Proteína)/química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Cisteína Proteases , Epitopos/química , Epitopos/imunologia , Epitopos/metabolismo , Haplorrinos , Células HeLa , Humanos , Proteína 2 de Membrana Associada ao Lisossomo , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Alinhamento de Sequência , Vesículas Transportadoras/química , Vesículas Transportadoras/imunologia , Trypanosoma cruzi/citologia , Trypanosoma cruzi/imunologia , Tubulina (Proteína)/imunologia , Tubulina (Proteína)/metabolismoRESUMO
Gangliosides are glycolipids mainly present at the plasma membrane (PM). Antibodies to gangliosides have been associated with a wide range of neuropathy syndromes. Particularly, antibodies to GM1 ganglioside are present in patients with Guillain-Barré syndrome (GBS). We investigated the binding and intracellular fate of antibody to GM1 obtained from rabbits with experimental GBS in comparison with the transport of cholera toxin (CTx), which binds with high affinity to GM1. We demonstrated that antibody to GM1 is rapidly and specifically endocytosed in CHO-K1 cells. After internalization, the antibody transited sorting endosomes to accumulate at the recycling endosome. Endocytosed antibody to GM1 is recycled back to the PM and released into the culture medium. In CHO-K1 cells, antibody to GM1 colocalized with co-endocytosed CTx at early and recycling endosomes, but not in Golgi complex and endoplasmic reticulum, where CTx was also located. Antibody to GM1, in contraposition to CTx, showed a reduced internalization to recycling endosomes in COS-7 cells and neural cell lines SH-SY5Y and Neuro2A. Results from photobleaching studies revealed differences in the lateral mobility of antibody to GM1 in the PM of analyzed cell lines, suggesting a relationship between the efficiency of endocytosis and lateral mobility of GM1 at the PM. Taken together, results indicate that two different ligands of GM1 ganglioside (antibody and CTx) are differentially endocytosed and trafficked, providing the basis to gain further insight into the mechanisms that operate in the intracellular trafficking of glycosphingolipid-binding toxins and pathological effects of neuropathy-associated antibodies.
Assuntos
Autoanticorpos/metabolismo , Toxina da Cólera/metabolismo , Células Epiteliais/metabolismo , Gangliosídeo G(M1)/metabolismo , Síndrome de Guillain-Barré/metabolismo , Vesículas Transportadoras/metabolismo , Animais , Autoanticorpos/imunologia , Autoanticorpos/farmacologia , Células CHO , Células COS , Chlorocebus aethiops , Toxina da Cólera/imunologia , Toxina da Cólera/farmacologia , Cricetinae , Cricetulus , Endocitose/imunologia , Células Epiteliais/imunologia , Gangliosídeo G(M1)/imunologia , Gangliosídeo G(M1)/farmacologia , Síndrome de Guillain-Barré/imunologia , Humanos , Transporte Proteico/imunologia , Coelhos , Vesículas Transportadoras/imunologiaRESUMO
Using confocal microscopy, MEST-1-positive immunofluorescence was observed within various Trypanosoma cruzi forms, except in cell-derived trypomastigotes. Glycosylinositol phosphorylceramides were identified by thin-layer chromatography immunostaining as the antigens recognized by MEST-1 in these parasites. In epimastigotes, labeling of MEST-1 coincided with acidic vesicles, indicating an internal localization of these glycoconjugates.