Lipid nanoparticle-mediated delivery of bta-miR-149-5p and bta-miR-1307 alleviates Mycoplasma bovis-induced mastitis via targeted regulation of macrophage inflammatory responses

Mycoplasma bovis ( M. bovis ) is a major causative agent of bovine mastitis, and increasing antibiotic resistance highlights the urgent need for alternative therapeutic strategies. Extracellular vesicles (EVs) serve as key mediators of host-pathogen communication by transferring regulatory microRNAs (miRNAs). In this study, we aimed to elucidate the functional roles of EV-associated miRNAs in M. bovis -induced inflammation and explore the therapeutic potential of lipid nanoparticle (LNP)-mediated miRNA delivery. We found that M. bovis infection remodeled the miRNA profile of EVs derived from bovine mammary epithelial cells (MAC-T), with a marked reduction in the abundance of bta-miR-149-5p and bta-miR-1307. Both bta-miR-149-5p and bta-miR-1307 were verified to negatively regulate M. bovis -induced inflammatory responses in BoMac cells. Mechanistically, bta-miR-149-5p directly targeted MAPK14 and suppressed the p38-MAPK/ATF2 signaling pathway, while bta-miR-1307 targeted MAP2K3 to inhibit M. bovis -induced inflammatory responses. Furthermore, co-overexpression of two miRNAs attenuated M. bovis -induced macrophage inflammatory responses, suggesting their potential combined regulatory effects. Additionally, we established a murine mastitis model and found Cy3-labeled, RGD-conjugated LNPs efficiently delivered bta-miR-149-5p and bta-miR-1307 mimics into mammary gland tissues. LNP-mediated co-delivery alleviated M. bovis -induced mastitis, as evidenced by reduced histopathological damage, decreased Ly6G-positive neutrophil infiltration, and reduced expression of pro-inflammatory cytokines. Collectively, this study reveals a novel EV-mediated miRNA regulatory axis utilized by M. bovis to facilitate macrophage inflammatory responses, wherein bta-miR-149-5p and bta-miR-1307 serve as critical anti-inflammatory modulators. LNP-mediated co-delivery of these miRNAs further offers a potential non-antibiotic therapeutic strategy to alleviate M. bovis -induced mastitis.