Personne :
Boilard, Éric

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Université Laval. Département microbiologie-infectiologie et d'immunologie
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Voici les éléments 1 - 5 sur 5
  • Publication
    Activated platelets can deliver mRNA regulatory Ago2•microRNA complexes to endothelial cells via microparticles
    (American Society of Hematology, 2013-07-11) Cloutier, Nathalie; Corduan, Aurélie; Duchez, Anne-Claire; Provost, Patrick; Laffont, Benoit; Plé, Hélène; Boilard, Éric
    Platelets play a crucial role in the maintenance of hemostasis, as well as in thrombosis. Upon activation, platelets release small membrane-bound microparticles (MPs) containing bioactive proteins and genetic materials from their parental cells that may be transferred to, and exert potent biological effects in, recipient cells of the circulatory system. Platelets have been shown to contain an abundant and diverse array of microRNAs, and platelet-derived MPs are the most abundant microvesicles in the circulation. Here we demonstrate that human platelets activated with thrombin preferentially release their miR-223 content in MPs. These MPs can be internalized by human umbilical vein endothelial cells (HUVEC), leading to the accumulation of platelet-derived miR-223. Platelet MPs contain functional Argonaute 2 (Ago2)•miR-223 complexes that are capable of regulating expression of a reporter gene in recipient HUVEC. Moreover, we demonstrate a role for platelet MP-derived miR-223 in the regulation of 2 endogenous endothelial genes, both at the messenger RNA and protein levels. Our results support a scenario by which platelet MPs may act as intercellular carriers of functional Ago2•microRNA complexes that may exert heterotypic regulation of gene expression in endothelial cells, and possibly other recipient cells of the circulatory system.
  • Publication
    Effects of pathogen reduction systems on platelet microRNAs, mRNAs, activation, and function
    (Informa Healthcare, 2019-04-18) Osman, Abdimajid; Corduan, Aurélie; Hitzler, Walter E; Provost, Patrick; Meyer, Claudius U.; Laffont, Benoit; Landry, Patricia; Boilard, Éric; Hellstern, Peter; Vamvakas, Eleftherios C.
    Pathogen reduction (PR) systems for platelets, based on chemically induced cross-linking and inactivation of nucleic acids, potentially prevent transfusion transmission of infectious agents, but can increase clinically significant bleeding in some clinical studies. Here, we documented the effects of PR systems on microRNA and mRNA levels of platelets stored in the blood bank, and assessed their impact on platelet activation and function. Unlike platelets subjected to gamma irradiation or stored in additive solution, platelets treated with Intercept (amotosalen + ultraviolet-A [UVA] light) exhibited significantly reduced levels of 6 of the 11 microRNAs, and 2 of the 3 anti-apoptotic mRNAs (Bcl-xl and Clusterin) that we monitored, compared with platelets stored in plasma. Mirasol (riboflavin + UVB light) treatment of platelets did not produce these effects. PR neither affected platelet microRNA synthesis or function nor induced cross-linking of microRNA-sized endogenous platelet RNA species. However, the reduction in the platelet microRNA levels induced by Intercept correlated with the platelet activation (p < 0.05) and an impaired platelet aggregation response to ADP (p < 0.05). These results suggest that Intercept treatment may induce platelet activation, resulting in the release of microRNAs and mRNAs from platelets. The clinical implications of this reduction in platelet nucleic acids secondary to Intercept remain to be established.
  • Publication
    Commercial dairy cow milk microRNAs resist digestion under simulated gastrointestinal tract conditions
    (Charles C. Thomas, Publisher, 2016-10-05) Gilbert, Caroline; Lee, Chan Ho C.; Savard, Patricia; Benmoussa, Abderrahim; Fliss, Ismaïl; Provost, Patrick; Laffont, Benoit; Laugier, Jonathan; Boilard, Éric
    Background: MicroRNAs are small, gene-regulatory noncoding RNA species present in large amounts in milk, where they seem to be protected against degradative conditions, presumably because of their association with exosomes. Objective: We monitored the relative stability of commercial dairy cow milk microRNAs during digestion and examined their associations with extracellular vesicles (EVs). Methods: We used a computer-controlled, in vitro, gastrointestinal model TNO intestinal model-1 (TIM-1) and analyzed, by quantitative polymerase chain reaction, the concentration of 2 microRNAs within gastrointestinal tract compartments at different points in time. EVs within TIM-1 digested and nondigested samples were studied by immunoblotting, dynamic light scattering, quantitative polymerase chain reaction, and density measurements. Results: A large quantity of dairy milk Bos taurus microRNA-223 (bta-miR-223) and bta-miR-125b (∼109–1010 copies/300 mL milk) withstood digestion under simulated gastrointestinal tract conditions, with the stomach causing the most important decrease in microRNA amounts. A large quantity of these 2 microRNAs (∼108–109 copies/300 mL milk) was detected in the upper small intestine compartments, which supports their potential bioaccessibility. A protocol optimized for the enrichment of dairy milk exosomes yielded a 100,000 × g pellet fraction that was positive for the exosomal markers tumor susceptibility gene-101 (TSG101), apoptosis-linked gene 2–interacting protein X (ALIX), and heat shock protein 70 (HSP70) and containing bta-miR-223 and bta-miR-125b. This approach, based on successive ultracentrifugation steps, also revealed the existence of ALIX−, HSP70−/low, and TSG101−/low EVs larger than exosomes and 2–6 times more enriched in bta-miR-223 and bta-miR-125b (P < 0.05). Conclusions: Our findings indicate that commercial dairy cow milk contains numerous microRNAs that can resist digestion and are associated mostly with ALIX−, HSP70−/low, and TSG101−/low EVs. Our results support the existence of interspecies transfer of microRNAs mediated by milk consumption and challenge our current view of exosomes as the sole carriers of milk-derived microRNAs.
  • Publication
    Platelet microparticles reprogram macrophage gene expression and function
    (Stuttgart Thieme, 2017-11-22) Corduan, Aurélie; Lee, Chan Ho C.; Duchez, Anne-Claire; Laffont, Benoit; Rousseau, Matthieu; Boilard, Éric
    Platelet microparticles (MPs) represent the most abundant MPs subtype in the circulation, and can mediate intercellular communication through delivery of bioactives molecules, such as cytokines, proteins, lipids and RNAs. Here, we show that platelet MPs can be internalised by primary human macrophages and deliver functional miR-126–3p. The increase in macrophage miR-126–3p levels was not prevented by actinomycin D, suggesting that it was not due to de novo gene transcription. Platelet MPs dose-dependently downregulated expression of four predicted mRNA targets of miR-126–3p, two of which were confirmed also at the protein level. The mRNA downregulatory effects of platelet MPs were abrogated by expression of a neutralising miR-126–3p sponge, implying the involvement of miR-126–3p. Transcriptome-wide, microarray analyses revealed that as many as 66 microRNAs and 653 additional RNAs were significantly and differentially expressed in macrophages upon exposure to platelet MPs. More specifically, platelet MPs induced an upregulation of 34 microRNAs and a concomitant downregulation of 367 RNAs, including mRNAs encoding for cytokines/chemokines CCL4, CSF1 and TNF. These changes were associated with reduced CCL4, CSF1 and TNF cytokine/chemokine release by macrophages, and accompanied by a marked increase in their phagocytic capacity. These findings demonstrate that platelet MPs can modify the transcriptome of macrophages, and reprogram their function towards a phagocytic phenotype
  • Publication
    Accès libre
    A subset of extracellular vesicles carries the bulk of microRNAs in commercial dairy cow’s milk
    (Co-Action Publishing, 2017-11-21) Gilbert, Caroline; Shan, Si Ting; Benmoussa, Abderrahim; Provost, Patrick; Ly, Sophia; Laugier, Jonathan; Boilard, Éric
    MicroRNAs are small gene-regulatory RNAs that are found in various biological fluids, including milk, where they are often contained inside extracellular vesicles (EVs), like exosomes. In a previous study, we reported that commercial dairy cow’s milk microRNAs resisted simulated digestion and were not exclusively associated with canonical exosomes. Here, we report the characterization of a milk EV subset that sediments at lower ultracentrifugation speeds and that contains the bulk of microRNAs. Milk EVs were isolated by differential ultracentrifugation and Iodixanol density gradient (IDG), and analysed for (1) microRNA enrichment by reverse transcription and quantitative polymerase chain reaction (RT-qPCR), and (2) EV-associated proteins by Western blot. Milk EVs were characterized further by dynamic light scattering (DLS), density measurements, fluorescent DiR and RNA labelling, high-sensitivity flow cytometry (HS-FCM), transmission electron microscopy (TEM), proteinase K and RNase A assay, and liquid chromatography tandem-mass spectrometry (LC-MS/MS). We found that the bulk of milk microRNAs (e.g., bta-miR-125b, bta-miR-148a, etc.) sediment at 12,000 g and 35,000 g. Their distribution pattern was different from that of exosome-enriched proteins, but similar to that of several proteins commonly found in milk fat globule membranes (MFGM), including xanthine dehydrogenase (XDH). These low-speed ultracentrifugation pellets contained cytoplasm-enclosing phospholipid bilayered membrane vesicles of a density comprised between 1.11 and 1.14 g/mL in Iodixanol. This milk EV subset of ~100 nm in diameter/~200 nm hydrodynamic size resisted to proteinase K digestion and protected their microRNA content from RNase A digestion. Our results support the existence of a milk EV subset pelleting at low ultracentrifugations speeds, with a protein coating comparable with MFGM, which contains and protects the bulk of milk microRNAs from degradation. This milk EV subset may represent a new EV population of interest, whose content in microRNAs and proteins supports its potential bioactivity