Commercial Dairy Cow Milk microRNAs Resist Digestion under Simulated Gastrointestinal Tract Conditions.

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.

Platelet microparticles reprogram macrophage gene expression and function.

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.