Profiling of the Exosomal Cargo of Bovine Milk Reveals the Presence of Immune‐ and Growth‐modulatory ncRNAs
Authors: Testroet Eric D. , Shome Sayane , Testroet Amber , Reecy James , Jernigan Robert L. , Zhu Meijun , Du Min , Clark Stephanie , Beitz Donald 
Published: June 2021

Bovine milk contains exosomes that carry a variety of non‐coding RNA (ncRNA). Exosomes play a role in intracellular communication and act in a paracrine or exocrine manner. Their properties protect the cargo against degradation. In vitro studies have demonstrated that gut epithelial cells can absorb exosomes and their cargo. It follows that exosomal ncRNA may play a role in mammary gland immune response of the cow and in the gut development of the pre‐weaned calf. We hypothesized that raw bovine milk would contain immune‐ and development‐related ncRNA. Our objective was to purify exosomes from raw milk and profile ncRNA by using next‐generation sequencing.Four replicates of raw milk were collected from a commercial dairy processing facility. Exosomes were isolated and total RNA was and profiled by Illumina RNA‐seq. MicroRNA (miRNA) was mapped to miRbase, normalized, and filtered. Panther gene ontology (GO) analysis on short‐listed target genes identified associated biological processes. Of 90 target genes in bovine (Bos taurus), 36 had hits with developmental process with mean fold‐enrichment (FE) value of 2.05. Of those genes, 14 associated with defense response, 18 with immune response, and 24 with immune system process were overrepresented in the query set (FE of 4.17, 5.43 and 3.94, respectively). The most highly represented miRNA was mir‐125a which is involved in host bacterial and viral immune response. Many genes had hits with GO biological processes related to cytokine response, MAPK pathway, leukocyte activation, and developmental processes, which support our hypothesis. An interaction network of 18 genes associated with immune response were derived via Genemania which predicted 18 additional closely regulated genes generating a gene‐interaction network with clustering coefficient of 0.473.Because ncRNAs are highly conserved, both mouse and human databases were probed that identified 5,015 target genes specific to human (Homo sapiens); 1,344 of those genes were related to development and were over‐represented with an FE of 1.18, consistent with support of mammary regeneration as well as gut epithelial development. Genes related to cellular pathways such as TGF‐β receptor signaling pathway (99 genes) and the Wnt signaling pathway (131 genes) were also cataloged. For 146 out of 414 genes in the query set from mouse (Mus musculus), specific target genes associated with anatomical structure development were over‐represented with an FE of 1.65, indicating the potential for these ncRNA to support mammary health and growth as well as pre‐weaned calf gut maturation.Of the 88 piwi‐interacting RNA (piRNA) found, 37 had annotations in pirBASE, although their function remains unclear. Furthermore, 305 instances of other RNA were found. By using the Ensembl database for identification, these transcripts were composed of 64 antisense RNA, 105 long‐intergenic ncRNA, 11 protein‐coding, and 50 processed transcripts, indicating a need for further exploration of possible roles of “other” RNAs contained in bovine exosomes. Together, our results support our hypothesis that exosomes of bovine milk contain ncRNA associated with immune and developmental functions.Support or Funding InformationThe research reported herein was generously supported by the Midwest Dairy Association.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.