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1.
Sci Rep ; 10(1): 19603, 2020 11 11.
Article in English | MEDLINE | ID: mdl-33177532

ABSTRACT

Neutrophils release extracellular traps (NETs) after interaction with microorganisms and physiological or synthetic products. NETs consist of decondensed chromatin complexed with proteins, some of them with microbicidal properties. Because NETs can modulate the functioning of HIV-1 target cells, we aimed to verify whether they modify HIV-1 replication in macrophages. We found that exposure of HIV-1-infected macrophages to NETs resulted in significant inhibition of viral replication. The NET anti-HIV-1 action was independent of other soluble factors released by the activated neutrophils, but otherwise dependent on the molecular integrity of NETs, since NET-treatment with protease or DNase abolished this effect. NETs induced macrophage production of the anti-HIV-1 ß-chemokines Rantes and MIP-1ß, and reduced the levels of integrated HIV-1 DNA in the macrophage genome, which may explain the decreased virus production by infected macrophages. Moreover, the residual virions released by NET-treated HIV-1-infected macrophages lost infectivity. In addition, elevated levels of DNA-elastase complexes were detected in the plasma from HIV-1-infected individuals, and neutrophils from these patients released NETs, which also inhibited HIV-1 replication in in vitro infected macrophages. Our results reveal that NETs may function as an innate immunity mechanism able to restrain HIV-1 production in macrophages.


Subject(s)
Extracellular Traps , HIV Infections/blood , HIV-1/physiology , Macrophages/virology , Neutrophils/cytology , Cell Survival , Cells, Cultured , Chemokines, CC/metabolism , DNA, Viral/metabolism , Extracellular Traps/genetics , HIV Infections/virology , HIV-1/pathogenicity , Humans , Macrophages/metabolism , Neutrophils/virology , Virus Replication/physiology
2.
PLoS One ; 15(8): e0237795, 2020.
Article in English | MEDLINE | ID: mdl-32833989

ABSTRACT

Extracellular vesicles (EVs) are small membrane-limited structures derived from outward budding of the plasma membrane or endosomal system that participate in cellular communication processes through the transport of bioactive molecules to recipient cells. To date, there are no published methodological works showing step-by-step the isolation, characterization and internalization of small EVs secreted by human primary macrophages derived from circulating monocytes (MDM-derived sEVs). Thus, here we aimed to provide an alternative protocol based on differential ultracentrifugation (dUC) to describe small EVs (sEVs) from these cells. Monocyte-derived macrophages were cultured in EV-free medium during 24, 48 or 72 h and, then, EVs were isolated from culture supernatants by (dUC). Macrophages secreted a large amount of sEVs in the first 24 h, with size ranging from 40-150 nm, peaking at 105 nm, as evaluated by nanoparticle tracking analysis and scanning electron microscopy. The markers Alix, CD63 and CD81 were detected by immunoblotting in EV samples, and the co-localization of CD63 and CD81 after sucrose density gradient ultracentrifugation (S-DGUC) indicated the presence of sEVs from late endosomal origin. Confocal fluorescence revealed that the sEVs were internalized by primary macrophages after three hours of co-culture. The methodology here applied aims to contribute for enhancing reproducibility between the limited number of available protocols for the isolation and characterization of MDM-derived sEVs, thus providing basic knowledge in the area of EV methods that can be useful for those investigators working with sEVs released by human primary macrophages derived from circulating monocytes.


Subject(s)
Cell Communication , Extracellular Vesicles/metabolism , Macrophages/metabolism , Blood Buffy Coat/cytology , Cell Differentiation , Cell Fractionation/methods , Centrifugation, Density Gradient/methods , Coculture Techniques , Healthy Volunteers , Humans , Intravital Microscopy , Macrophages/cytology , Macrophages/ultrastructure , Microscopy, Confocal , Microscopy, Electron, Scanning , Microscopy, Fluorescence , Monocytes/physiology , Primary Cell Culture
3.
Sci Rep ; 10(1): 2715, 2020 02 17.
Article in English | MEDLINE | ID: mdl-32066757

ABSTRACT

Neutrophil extracellular traps (NETs) emerge from the cell as a DNA scaffold associated with cytoplasmic and granular proteins, able to immobilize and kill pathogens. This association occurs following nuclear and granular membrane disintegration, allowing contact with the decondensed chromatin. Thus, it is reasonable to speculate that the DNA can also mix with miRNAs and carry them in NETs. Here, we report for the first time the presence of the miRNA carriers associated with NETs and miRNAs present in NET-enriched supernatants (NET-miRs), thus adding a novel class of molecules and new proteins that can be released and transported in the NET platform. We observed that the majority of NET-miRs were common to all four stimuli used (PMA, interleukin-8, amyloid fibrils and Leishmania), and that miRNA-142-3p carried by NETs down-modulates protein kinase Cα and regulates TNF-α production in macrophages upon NET interaction with these cells. Our findings unveil a novel role for NETs in the cell communication processes, allowing the conveyance of miRNA from neutrophils to neighboring cells.


Subject(s)
Cell Communication/immunology , Extracellular Traps/immunology , MicroRNAs/genetics , Neutrophils/immunology , Tumor Necrosis Factor-alpha/genetics , Amyloid/pharmacology , Antagomirs/genetics , Antagomirs/metabolism , Culture Media, Conditioned/pharmacology , Extracellular Traps/metabolism , Gene Expression Regulation , Humans , Interleukin-8/pharmacology , Leishmania braziliensis , MicroRNAs/antagonists & inhibitors , MicroRNAs/immunology , Neutrophils/drug effects , Neutrophils/microbiology , Primary Cell Culture , Protein Kinase C-alpha/genetics , Protein Kinase C-alpha/immunology , Signal Transduction , THP-1 Cells , Tetradecanoylphorbol Acetate/pharmacology , Tumor Necrosis Factor-alpha/immunology
4.
Eur J Cell Biol ; 96(2): 131-142, 2017 Mar.
Article in English | MEDLINE | ID: mdl-28236495

ABSTRACT

Giardia intestinalis (G.I), is an anaerobic protozoan and the aetiological agent of giardiasis, a diarrhoea present worldwide and associated with poverty. G.I has a simple life cycle alternating between cyst and trophozoite. Cysts are transmitted orally to the stomach and transform to trophozoites in the intestine by a multifactorial process. Recently, microvesicles (MVs) have been found to be released from a wide range of eukaryotic cells. We have observed a release of MVs during the life cycle of G.I., identifying MVs from active trophozoites and from trophozoites differentiating to the cyst form. The aim of the current work was to investigate the role of MVs from G.I in the pathogenesis of giardiasis. MVs from log phase were able to increase the attachment of G. intestinalis trophozoites to Caco-2 cells. Moreover, MVs from G. intestinalis could be captured by human immature dendritic cells, resulting in increased activation and allostimulation of human dendritic cells. Lipid rafts participate in the MV biogenesis and in the attachment to Caco-2 cells. Nevertheless, proteomic analysis from two types of MVs has shown slight differences at the protein levels. An understanding of biogenesis and content of MVs derived from trophozoites might have important implications in the pathogenesis of the disease.


Subject(s)
Cell-Derived Microparticles/immunology , Giardia lamblia/immunology , Giardiasis/immunology , Animals , Caco-2 Cells , Extracellular Vesicles/immunology , Host-Pathogen Interactions/immunology , Humans , Immunity, Innate
5.
FEBS Lett ; 588(6): 956-61, 2014 Mar 18.
Article in English | MEDLINE | ID: mdl-24560788

ABSTRACT

To produce an infection Trypanosoma cruzi must evade lysis by the complement system. During early stages of infection, the lectin pathway plays an important role in host defense and can be activated by binding of mannan-binding lectin (MBL) to carbohydrates on the surface of pathogens. We hypothesized that MBL has a dual role during parasite-host cell interaction as lectin complement pathway activator and as binding molecule to invade the host cell. We used two polarized strains of T. cruzi, R4 (susceptible) and 860 (resistant) strains, to investigate the role of MBL in complement-mediated lysis. Interestingly R4, but not 860 metacyclic strain, markedly increases the invasion of host cells, suggesting that MBL drives the invasion process while the parasite deactivates the Lectin complement pathway.


Subject(s)
Mannose-Binding Lectin/physiology , Protozoan Proteins/physiology , Trypanosoma cruzi/physiology , Animals , Chagas Disease/parasitology , Chlorocebus aethiops , Complement Activation , Complement Pathway, Alternative , Host-Parasite Interactions , Humans , Immunity, Innate , Vero Cells
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