Bovine milk-derived exosomes from colostrum are enriched with proteins implicated in immune response and growth

Abstract

Exosomes are extracellular vesicles secreted by multiple cell types into the extracellular space. They contain cell-state specific cargos which often reflects the (patho)physiological condition of the cells/organism. Milk contains high amounts of exosomes and it is unclear whether their cargo is altered based on the lactation stage of the organism. Here, we isolated exosomes from bovine milk that were obtained at various stages of lactation and examined the content by quantitative proteomics. Exosomes were isolated by OptiPrep density gradient centrifugation from milk obtained from cow after 24, 48 and 72 h post calving. As control, exosomes were also isolated from cows during mid-lactation period which has been referred to as mature milk (MM). Biochemical and biophysical characterization of exosomes revealed the high abundance of exosomes in colostrum and MM samples. Quantitative proteomics analysis highlighted the change in the proteomic cargo of exosomes based on the lactation state of the cow. Functional enrichment analysis revealed that exosomes from colostrum are significantly enriched with proteins that can potentially regulate the immune response and growth. This study highlights the importance of exosomes in colostrum and hence opens up new avenues to exploit these vesicles in the regulation of the immune response and growth.

Milk MicroRNAs in Health and Disease

“…Isolation and purification of milk EVs through different methodologies (ultracentrifugation, size-exclusion chromatography, and exoquick) allowed to discriminate between at least two EV subsets in milk including exosomes, pelleting at 100,000 g, and larger and denser EVs sedimenting at lower ultracentrifugation speeds (Benmoussa et al, 2017(Benmoussa et al, , 2018Théry et al, 2018;van Herwijnen et al, 2016;Wang, 2017). Proteomic analysis of these EVs in humans, cows, and pigs (Benmoussa et al, 2018;Reinhardt, Lippolis, Nonnecke, & Sacco, 2012;Samuel et al, 2017) suggest that the most abundant proteins originate from mammary gland cells (for example, XDH, MFGE-8, BTN1H1), but numerous proteins specific to milk EVs were associated with immune cells (Benmoussa et al, 2018;Reinhardt et al, 2012;van Herwijnen et al, 2016). Other studies supported the specific origin of milk exosomes from breast cell lines (Larssen et al, 2017).…”

Identification of protein markers for extracellular vesicle (EV) subsets in cow's milk

“…We assessed whether widely accepted EV protein markers [21,34] could be used to discriminate the nature and subsets of the EVs isolated from commercial cow milk. Two previous studies, performed on milk or another biological fluid (e.g.…”

“…cell culture supernatant), suggested several protein markers for different EV subsets, based either on isolation procedures and physical properties of the EVs (light, dense, large or multiple EVs) [21], or on cellular origin of the proteins associated with those EVs -proteins from the endosomal sorting complexes required for transport (ESCRT) and proteins from the Rab family strongly associated to MVB-derived EVs (i.e. exosomes) [34]. The concomitant presence of the three tetraspanins CD9, CD63 and CD81 was also suggested to be primordial to define an EV subset as exosomes [21].…”

Arrestin‐Domain Containing Protein 1 (Arrdc1) Regulates the Protein Cargo and Release of Extracellular Vesicles

“…Next, equal protein amount (30 μg) of the WCL, ectosomes, and exosomes fraction of WT and Arrdc1 −/− MEFs were subjected to label‐free quantitative proteomics analysis as described previously . Briefly, proteins were separated by 1D‐SDS PAGE, gel bands were excised, reduced, alkylated and subjected to trypsin digestion.…”