Personne : Gilbert, Caroline
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Université Laval. Département de microbiologie-infectiologie et d'immunologie
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- PublicationRestreintCommercial 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, ÉricBackground: 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.
- PublicationAccès libreA 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, ÉricMicroRNAs 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
- PublicationAccès libreConcentrates of two subsets of extracellular vesicles from cow’s milk modulate symptoms and inflammation in experimental colitis(Nature Publishing Group, 2019-10-10) Gilbert, Caroline; Sévigny, Jean; Salem, Mabrouka; Benmoussa, Abderrahim; Michel, Sara; Provost, Patrick; Diallo, IdrissaExtracellular vesicles (EVs) are involved in cell-to-cell communication and modulation of numerous physiological and pathological processes. EVs are found in large quantities in milk and contain several inflammation- and immunity-modulating proteins and microRNAs, through which they exert beneficial effects in several inflammatory disease models. Here, we investigated the effects of two EV subsets, concentrated from commercial cow’s milk, on a murine model of colitis induced with dextran sodium sulfate (DSS). P35K EVs, isolated by ultracentrifugation at 35,000 g, and P100K EVs, isolated at 100,000 g, were previously characterized and administered by gavage to healthy and DSS-treated mice. P35K EVs and, to a lesser extent, P100K EVs improved several outcomes associated to DSS-induced colitis, modulated the gut microbiota, restored intestinal impermeability and replenished mucin secretion. Also, P35K EVs modulated innate immunity, while P100K EVs decreased inflammation through the downregulation of colitis-associated microRNAs, especially miR-125b, associated with a higher expression of the NFκB inhibitor TNFAIP3 (A20). These results suggest that different milk EV subsets may improve colitis outcomes through different, and possibly complementary, mechanisms. Further unveiling of these mechanisms might offer new opportunities for improving the life of patients with colitis and be of importance for milk processing, infant milk formulation and general public health.
- PublicationAccès libreIdentification of protein markers for extracellular vesicle (EV) subsets in cow’s milk(Elsevier BV, 2018-08-25) Bourassa, Sylvie; Gilbert, Caroline; Benmoussa, Abderrahim; Provost, Patrick; Gotti, ClarisseExtracellular vesicles (EVs), like exosomes, are small membrane vesicles involved in cell-to-cell communications that modulate numerous biological processes. We previously discovered a new EV subset in milk (sedimenting at 35,000 g; 35 K) that protected its cargo (RNAs and proteins) during simulated digestion and was more enriched in microRNAs than exosomes (sedimenting at 100 K). Here, we used LC-MS/MS to push further the comparison between these two pellets. Commonly used EV markers were not differentially enriched between the pellets, questioning their use with cow's milk EVs. Similarly, the majority of the quantified proteins were equally enriched between the two pellets. Nevertheless, 20 proteins were specific to 35 K, while 41 were specifically enriched in 100 K (p < 0.05), suggesting their potential use as specific markers. Loaded with these proteins, the EVs in these pellets might regulate translation, proliferation and cell survival for 35 K, and metabolism, extracellular matrix turnover and immunity for 100 K. This approach also brought new insights into milk EV-associated integrins and their possible role in specifically targeting recipient cell types. These findings may help better discriminate between milk EVs, improve our understanding of milk EV-associated protein function and their possible use as therapeutic tools for the management of immunity- and metabolism-associated disorders.