The Protective Role of Exosome-Derived MicroRNAs and Proteins from Human Breast Milk against Infectious Agents

Abstract

Human breast milk (HBM)-derived exosomes contain various biological and immunological components. However, comprehensive immune-related and antimicrobial factor analysis requires transcriptomic, proteomic, and multiple databases for functional analyses, and has yet to be conducted. Therefore, we isolated and confirmed HBM-derived exosomes by detecting specific markers and examining their morphology using western blot and transmission electron microscopy. Moreover, we implemented small RNA sequencing and liquid chromatography-mass spectrometry to investigate substances within the HBM-derived exosomes and their roles in combating pathogenic effects, identifying 208 miRNAs and 377 proteins associated with immunological pathways and diseases. Integrated omics analyses identified a connection between the exosomal substances and microbial infections. In addition, gene ontology and the Kyoto Encyclopedia of Genes and Genomes pathway analyses demonstrated that HBM-derived exosomal miRNA and proteins influence immune-related functions and pathogenic infections. Finally, protein-protein interaction analysis identified three primary proteins (ICAM1, TLR2, and FN1) associated with microbial infections mediating pro-inflammation, controlling infection, and facilitating microbial elimination. Our findings determine that HBM-derived exosomes modulate the immune system and could offer therapeutic strategies for regulating pathogenic microbial infection.

Keywords: exosome; human breast milk; microbial defense; proteomics; small RNA sequencing.

Figure 1

Summary of HBM-derived exosome. (A) HBM-derived exosome isolation through ultracentrifugation and SEC (n = 10). First, 400 mL HBMs were centrifuged at 2000× g, and the upper layer (milk fat) and pellet (cell and debris) were discarded. The middle section (whey) was used, and 3 mL was the final crude exosome sample volume. Exosomes were resuspended and separated into 15 fractions relative to protein size using the SEC (43.92 ± 4.28 μg/μL, n = 11), and Fractions 7–9 were selected. (B) HBM-derived exosomes were characterized by specific (CD81, CD9, Annexin V, CD54/ICAM1, Flotillin-1, and Alix) and non-specific (GM130) exosomal markers through Western blot analysis. (C) Transmission electron microscopy (TEM) was used to identify exosome morphology and size (scale bar: 200 nm). (D) Nanoparticle tracking analysis (NTA) was performed to determine the size distribution and concentration of the exosomes.

Figure 2

Small RNA sequencing and expression profiles of HBM-derived exosomes. (A) Three different HBM-derived exosome samples were assessed for migration and peak patterns for quality control before small RNA sequencing. (B,C) Sixty miRNAs, consistently expressed in all three samples of HBM-derived exosome, were selected (**, p < 0.005; the p-value was demonstrated through correlation analysis), and their expression levels were presented as normalized data (Log2; ND).

Figure 3

GO annotation analysis of HBM-derived exosomal miRNAs. (A) HBM-derived exosomal miRNA cellular components (CC), molecular functions (MF), and biological processes (BP) were classified through GO annotation analysis (p < 0.05) (B) Based on the GO:BP analysis, immune-related BPs associated with HBM-derived exosomal miRNAs were determined. (C) A total of 45 terms were identified, and miRNA quantities associated with specific GO terms were ranked by p-values using the Hyper-geometric test (Protein quantity: blue bar; −Log10 p-value: blue line; p = 0.05: red line).

Figure 4

GO annotation analysis of HBM-derived exosomal proteins. (A) HBM-derived exosomal protein cellular components (CC), molecular functions (MF), and biological processes (BP) were categorized through GO annotation analysis (p < 0.05). (B) A total of 31 immune-related BPs of HBM-derived exosomal proteins were identified through GO:BP analysis. (C) Protein quantities associated with specific GO terms were ranked by p-values and visualized with Hyper-geometric test p-values (Protein quantity: blue bar; −Log10 p-value: blue line; p-value = 0.05: red line).

Figure 5

Pathogenic microbial infection protein-protein interactions. (A) All proteins identified were associated with immune-related proteins determined by the KEGG pathway analysis. Microbial infection includes three categories: bacterial (n = 32), viral (n = 16), and protozoal (n = 8) (B) Pathogen-related proteins displayed protein-protein interactions (red: bacterial, blue: viral, green: protozoal, purple: bacterial and viral, yellow-green: bacterial and protozoal, cyan: viral and protozoal, black: all three microbial infections, interactor = 0, confidence score = 0.900).