| Popis: |
In recent years, several studies demonstrated the role of exosomes in intercellular communications. Specialized secretion systems are used by numerous bacterial pathogens to export virulence factors into host target cells. Leishmania deliver effectors proteins into host cells; however, the mechanisms involved have remained elusive. The aim of this study was to isolate, characterize Leishmania infantum exosomes and to investigate the biological activity of exosomes in macrophage cultures.Human cell lines used were U937 cells and macrophages derived from U937 after treatment with 25 ng/ml PMA.Western blot assay used antibodies to HSP70, HSP83/90. Exosomes were collected by L. infantum -conditioned medium by ultracentrifugation.IL10, IFN?, IL1?IL1?, IL12, IL4 and IL18 secretion were evaluated by ELISA kit.We first purified extracellular vesicles shed by L. Infantum promastigotes and amastigotes on a sucrose gradient and later we characterized these extracellular vesicles for HSP70, HSP83/90 and acetylcholinesterase. Furthermore we performed a NanoSight nanoparticle tracking analysis revealed an average of the mode value of 76 ± 5 nm for promastigotes and 94± 5 nm for amastigotes exosomes. All these data demonstrated that L. Infantum released exosomes. The treatment of U937 cells with 10 μg/ml of promastigote and amastigote exosomes showed an increase in motility of these cells that can facilitate the progression of infection. We showed also an overproduction of IL10 by macrophages after treatment with exosomes that support parasite persistence and disease establishment, while exosomes limited the production of IFN-? and IL12 that block the parasite killing and host protection, as well as the production of IL1?, IL1? and IL4. Regarding IL18 production,that is involved in the Th1-type immune responses,L. exosomes determined a reduction in the production of IL18 in U937 monocyte culturescompared to control. Value that does not vary instead macrophages cultures. |
