Milk represents a complex pool of nutrients and bioactive components that are indispensable for the growth and development of the infant. The key well‐established roles played by milk's bioactive components in the infant are those at the level of the infant's intestinal and immune development. Through its bioactive components, including proteins, lipids, and oligosaccharides, milk helps the infant develop a mature intestinal identity with fully active digestive, absorptive, and barrier capacity and shapes both innate and adaptive immune responses. Recent evidence points to a new class of milk bioactive components including milk extracellular vesicles (EVs) and microRNAs, which are hypothesized to take part in contributing to infant development. The potential functions of milk microRNAs and EVs in the consumer's systems are not limited to the infant as these components can also be found in bovine milk, both raw and processed. Hence, adult consumers could also be influenced by milk EVs and microRNAs, which could affect their intestinal homeostasis particularly under pathological conditions. Nonetheless, the debate regarding the stability of milk EVs and microRNAs in the digestive tract persists, and their bioavailability and bioactivity in the consumer's tissues are still arguable. In this review, we discuss the potential functions mediated by milk microRNAs and EVs in the epithelial and immune components as well as the microbiome of the intestinal mucosa in health and disease. We also discuss the bioavailability, bioaccessibility, and bioactivity of milk EVs and microRNAs in consumer's tissues.

Ebola virus (EBOV) is a virulent pathogen, notorious for inducing life-threatening hemorrhagic fever, that has been responsible for several outbreaks in Africa and remains a public health threat. Yet, its pathogenesis is still not completely understood. Although there have been numerous studies on host transcriptional response to EBOV, with an emphasis on the clinical features, the impact of EBOV infection on post-transcriptional regulatory elements, such as microRNAs (miRNAs), remains largely unexplored. MiRNAs are involved in inflammation and immunity and are believed to be important modulators of the host response to viral infection. Here, we have used small RNA sequencing (sRNA-Seq), qPCR and functional analyses to obtain the first comparative miRNA transcriptome (miRNome) of a human liver cell line (Huh7) infected with one of the following three EBOV strains: Mayinga (responsible for the first Zaire outbreak in 1976), Makona (responsible for the West Africa outbreak in 2013–2016) and the epizootic Reston (presumably innocuous to humans). Our results highlight specific miRNA-based immunity pathways and substantial differences between the strains beyond their clinical manifestation and pathogenicity. These analyses shed new light into the molecular signature of liver cells upon EBOV infection and reveal new insights into miRNA-based virus attack and host defense strategy.

MicroRNAs (miRNAs) are important gene regulatory molecules involved in a broad range of cellular activities. Although the existence and functions of miRNAs are clearly defined and well established in eukaryotes, this is not always the case for those of viral origin. Indeed, the existence of viral miRNAs is the subject of intense controversy, especially those of RNA viruses. Here, we characterized the miRNA transcriptome of cultured human liver cells infected or not with either of the two Ebola virus (EBOV) variants: Mayinga or Makona; or with Reston virus (RESTV). Bioinformatic analyses revealed the presence of two EBOV-encoded miRNAs, miR-MAY-251 and miR-MAK-403, originating from the EBOV Mayinga and Makona variants, respectively. From the miRDB database, miR-MAY-251 and miR-MAK-403 displayed on average more than 700 potential human host target candidates, 25% of which had a confidence score higher than 80%. By RT-qPCR and dual luciferase assays, we assessed the potential regulatory effect of these two EBOV miRNAs on selected host mRNA targets. Further analysis of Panther pathways unveiled that these two EBOV miRNAs, in addition to general regulatory functions, can potentially target genes involved in the hemorrhagic phenotype, regulation of viral replication and modulation of host immune defense.

Milk microRNAs are believed to have regulatory functions in the consumer's cells. Milk from different species is enriched in microRNAs predicted to influence immunity, metabolism, and intestinal homeostasis. For milk microRNAs to regulate gene expression in the consumer, they must survive digestion and be present at sufficient levels to influence intestinal cells and potentially beyond intestinal cells. Milk microRNAs are proposed to be protected from degradation through their association with milk extracellular vesicles (EVs), which might also deliver them to cells. Studies on milk microRNA oral transfer and tissue bioavailability are limited by interspecies sequence homology, making it difficult to distinguish endogenous from exogenous microRNAs. Here, we used a transgenic (TG) cow model expressing four unique microRNA sequences (AmiRs) in its milk to study their association with milk EVs, their resistance to in vitro digestion, and AmiR uptake and regulatory activity in vitro. We confirmed the presence of the four milk EV populations in raw wild‐type (WT) and TG cow milk, similar to those previously reported in commercial (pasteurized) cow milk, and confirmed their association with AmiRs and classical milk microRNAs. AmiRs showed differential resistance to simulated adult digestion. No statistically significant regulatory effect of AmiRs was observed in Caco‐2 cells exposed to TG milk EVs, suggesting absent or non‐functional cellular uptake. The intent of using this model was to perform in vitro analysis to lay the groundwork for later in vivo bioavailability studies, taking advantage of the uniqueness of the AmiR sequences and bypassing the limitation of microRNA sequence homology.

Mother’s milk is known for its crucial roles in infant health and development. Probably the most commonly studied effects of milk are those on an infant’s intestinal barrier, metabolism, and immunity. While these functions of milk were mostly attributed to its protein and fat content, recent evidence points to a potential role of milk microRNAs in these processes. MicroRNAs are small non-coding RNAs that can fine-tune gene expression at the post-transcriptional level. Human milk (HM) is rich in microRNAs, which are mainly found associated with milk extracellular vesicles (EVs). HM microRNAs are proposed to transfer from mother to infant via breastfeeding and execute gene regulatory functions in infant cells. For microRNAs to be able to act as “genetic programmers” rather than mere nutritional molecules, they should resist digestion in the infant’s gastrointestinal tract. Milk EVs are believed to protect microRNAs against degradation and facilitate their delivery to the cells. Here, we used two lots of pasteurized HM that were originally destined for human milk banks. We showed that HM contains different populations of EVs with different physicochemical properties, similar to those previously identified in commercial bovine milk. We also showed that these EVs, which are often discarded, contain the majority of HM microRNAs. Finally, we showed that three highly abundant milk microRNAs resisted differentially to infant’s simulated digestion conditions, with a relatively small number of microRNAs surviving a two-hour digestion. Milk microRNA copy numbers surviving digestion may be too low to influence gene expression in infant cells. Highlights: Pasteurized HM contains heterogeneous populations of EVs. These EVs associate with the majority of HM microRNAs. Different microRNAs show varying stability during infant digestion. The copy number of milk miR-148a-3p surviving digestion might be too low to influence gene expression in infant cells.

Milk microRNAs are believed to play gene regulatory functions in the consumer's cells. Milk from different species is enriched in microRNAs predicted to influence immunity, metabolism, and intestinal homeostasis. For milk microRNAs to regulate gene expression in the consumer, they must survive digestion and be present at sufficient levels to influence intestinal cells and potentially beyond-intestinal cells. Milk microRNAs are proposed to be protected from degradation through their association with milk extracellular vesicles (EVs), which might also deliver them to cells. Studies on milk microRNA oral transfer and tissue bioavailability are limited by interspecies sequence homology, making it difficult to distinguish endogenous from exogenous microRNAs. Here, we used a transgenic (TG) cow model expressing four unique microRNA sequences (AmiRs) in its milk to study their association with milk EVs, their resistance to in vitro digestion, and AmiR uptake and regulatory activity in vitro. We confirmed the presence of the four milk EV populations in raw wild-type (WT) and TG cow milk, similar to those previously reported in commercial (pasteurized) cow milk, and confirmed their association with AmiRs and classical milk microRNAs. AmiRs showed differential resistance to simulated adult digestion. In vitro uptake studies showed a modest gene regulatory effect of AmiRs in Caco-2 cells incubated with TG milk EVs. The intent of using this model was to perform in vitro analysis which could lay the groundwork for later in vivo bioavailability studies, taking advantage of the uniqueness of the AmiRs sequences and bypassing the limitation of microRNA sequence homology.