Abstract: The aim of this study was to investigate the effects of exosomes (Exo) from different milk sources on macrophage polarization. Ten milk samples were collected aseptically and the properties of the milk samples were determined by somatic cell count (SCC) and bacteriological identification; the bovine milk-derived exosomes were enriched and purified by differential ultracentrifugation; exosomes were identified by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and western blot (WB); the fluorescent tracer method was used to detect whether exosomes could be internalized by macrophages (Raw264.7); real-time fluorescent quantitative PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to detect changes in mRNA levels of pro-inflammatory cytokines, anti-inflammatory cytokines and protein levels after the action of bovine milk-derived exosomes on Raw264.7 cells. According to SCC, there were 4 normal milk samples and 6 inflammatory milk samples. Four strains of pathogenic bacteria were isolated from milk samples of inflamed cows and their biological characteristics were consistent with Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae respectively; the exosomes were separated by ultracentrifugation and different bovine milk-derived exosomes were internalized by Raw264.7 after co-culture with it for 6 h. Compared with the control group (Con), compared with the control (Con) group, both the low somatic cell count source exosomes (L-Exo) and high somatic cell count source exosomes (H-Exo) groups significantly increased Raw264.7 cell tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 mRNA and protein expression levels (P<0.05), nitric oxide synthase (iNOS) and macrophage mannose receptor CD206 protein expression were significantly increased (P<0.05), IL-10, transforming growth factor (TGF)-β mRNA and protein levels were decreased (P<0.05), arginase (Arg)-1 mRNA expression levels were decreased (P<0.05). During the inflammatory response in the mammary gland of cows, mastitis bovine milk-derived exosomes can promote the polarisation of macrophages towards M1 type, express high levels of pro-inflammatory cytokines and promote the inflammatory response, while exosomes secreted in the healthy state of the mammary gland can mediate the polarisation of macrophages towards M2 type, express high levels of anti-inflammatory cytokines, participate in the immune regulation of the body and have certain anti-inflammatory effects, this demonstrates that milk-derived exosomes can influence the process of macrophage polarization.

Key words: bovine mastitis, exosomes, polarization of macrophages, lipopolysaccharide