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.

Figure 1

Western blot analysis of exosomes isolated from MM and colostrum. (a) Western blot analysis of fractions of increasing density obtained from the OptiPrep density gradient centrifugation which were probed for exosomal markers Alix and TSG101. TSG101 was identified in all the samples from fractions 5–9 corresponding to the buoyant density of 1.08–1.22 g/mL. The presence of Alix was limited to colostrum samples only. (b) Western blot analysis revalidating the presence of Alix in higher abundance only within colostrum exosomes as compared to mature milk (MM) and commercial milk (CM). TSG101 was found in all the samples including commercial milk exosomes. Exosomes isolated from LIM1215 colorectal cancer cells (LIM1215) was used as a positive control. GM130 was detected in LIM1215 colorectal cancer cell lysates (WCL) and was absent in the milk derived exosomal samples confirming the depletion of contaminating vesicles arising from apoptosis.

Figure 2

Biophysical characterization of exosomes isolated from MM and colostrum. (a) TEM images of exosomes isolated by OptiPrep density gradient centrifugation revealed vesicles with a morphology similar to exosomes and a diameter in the range of 30–150 nm for all four samples. The colostrum samples had meagrely scattered vesicles with higher protein background whereas the MM samples revealed a rich population of exosomes with typical cup-shaped morphology. (b) NTA revealed peak intensities to be 125, 95, 95 and 145 nm for 24, 48, 72 h colostrum and MM samples, respectively.

Figure 3

Proteomics analysis of exosomes isolated from MM and colostrum. (a) A four-way Venn diagram of proteins distributed between 24, 48, 72 h colostrum and MM exosome samples revealing 1372 proteins common to all dataset. (b) Heatmaps showing proteins that are 3-fold more abundant in exosomes isolated from 24 h colostrum samples compared to MM. A distinct pattern was revealed where proteins that were seen to be highly abundant in 24 h colostrum exosomes steadily diminished in 48 and 72 h colostrum exosomes and reached similar levels to the MM exosomes. (b) Proteins that are 3-fold more highly abundant in exosomes isolated from MM samples compared to 24 h colostrum sample were also plotted as a heatmap and revealed a similar distribution pattern.

Figure 4

Exosomes from bovine milk are enriched with exosomal markers. (a) A heatmap showing enrichment of exosomal marker proteins in colostrum and MM exosomes. The scaled expression of each protein, denoted as the row Z-score, is plotted in the red–blue colour scale. High expression levels are indicated in red and low expression levels are shown in blue. Similar to western blot analysis, Alix (PDCD6IP) was found to be of higher abundance in the colostrum samples when compared with the MM samples. The 72 h colostrum samples had a higher level of many of the ESCRT component proteins. (b) Higher abundance of Rab family proteins was observed in both colostrum and MM exosomes. (c) An enrichment of integrins, which play a crucial role in exosomal uptake by target cells was observed in all the exosomes.

Figure 5

Exosomes from colostrum are enriched with proteins implicated in immune response. (a) Venn diagram of proteins identified in MM and colostrum exosomes. More than 4980 proteins were uniquely identified in colostrum samples. (b) Functional enrichment analysis using FunRich revealed various proteins associated with innate immune response, inflammatory response, acute-phase response, platelet activation, and cell growth were highly enriched in exosomes isolated from colostrum (24, 48 and 72 h sample) along with significant enrichment in 24 h sample on its own. * Denotes P < 0.05. (c) Histogram representing proteins related to complement activation and antimicrobial peptides were significantly enriched in exosomes from colostrum and 24 h sample when compared with MM. * Denotes P < 0.05.