Comprehensive multiomic analysis of extracellular vesicles from Mycoplasma bovis-infected bovine mammary epithelial cells identifies proteins and miRNAs that induce inflammatory responses in macrophages

Mycoplasma bovis can lead to a decline in milk quality and yield, thereby causing significant economic losses worldwide. Extracellular vesicles (EVs) are crucial for triggering immune cell responses to infection. This study aimed to demonstrate the immunomodulatory effects of EVs released by bovine mammary epithelial cells (MAC-T cells) infected with M. bovis on bovine macrophages (BoMacs). After EVs were extracted from M. bovis-infected MAC-T cells (M. bovis NX2-EVs) as well as from uninfected MAC-T cells (Ctrl-EVs), they were incubated with BoMacs to assess their potential to induce cytokine expression. The results showed that M. bovis NX2-EV-treated BoMacs exhibited significantly increased expression of TNF-α, IL-1β, and IL-6. Additionally, the differentially expressed genes mainly involved the TNF, NF-kappa B and IL-17 signalling pathways, with endocytosis and megalocytosis recognized as the main pathways through which BoMacs can take up EVs. Furthermore, mass spectrometry and RNA-seq were used to determine the protein and miRNA expression profiles of Ctrl-EVs and M. bovis NX2-EVs. Overall, 27 And 86 proteins were significantly downregulated and upregulated, respectively, in M. bovis NX2-EVs compared with those in Ctrl-EVs. Similarly, a total of 9 miRNAs were upregulated, while 2 miRNAs were downregulated in M. bovis NX2-EVs. Finally, JCHAIN, MAPRE1, miR-1307, and miR-149-5p were identified as differentially expressed proteins and miRNAs in M. bovis NX2-EVs, thus highlighting their involvement in cellular immune regulation and related diseases. These results reveal the mechanism of host resistance to M. bovis infection and provide new insights for exploring the pathogenic mechanism of M. bovis.