Fenofibrate suppresses Mycoplasma bovis infection via autophagy-mediated cholesterol regulation in bovine mammary epithelial cells and murine mammary tissue.

Background: Mycoplasma bovis mastitis is an important disease of dairy cows; however, there are no commercial M. bovis vaccines and antimicrobial resistance is increasing. Furthermore, M. bovis lacks a cell wall and relies on host-derived cholesterol for survival and growth. Methods: We evaluated effects of fenofibrate, a peroxisome proliferator-activated receptor α (PPARα) agonist, on M. bovis infection, using both bovine mammary epithelial cells and a murine mastitis model. In vitro analyses assessed autophagy, nuclear Transcription Factor EB (TFEB) and Transcription Factor E3 (TFE3) translocation, cholesterol metabolism, and bacterial localization, whereas in vivo evaluations included inflammatory responses, lysosomal/autophagy protein expression, cholesterol content, and bacterial burden. Results: Fenofibrate enhanced autophagic flux via upregulation of LC3B and LAMP2, promoted nuclear translocation of TFEB and TFE3, and reduced intracellular cholesterol by repressing 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase (HMGCR) and Sterol Regulatory Element-Binding Transcription Factor 2 (SREBF2) while increasing Acetyl-Coenzyme A Acetyltransferase 1 (ACAT1) expression. Based on confocal imaging, fenofibrate disrupted co-localization between M. bovis and free cholesterol while enhancing its uptake by autophagosomes. In a murine mastitis model, fenofibrate alleviated inflammatory cell infiltration and cytokine release, restored lysosomal and autophagy protein expression, reduced cholesterol content, and significantly lowered bacterial burden. Conclusion: Fenofibrate enhanced defense capability of mammary epithelial cells against M. bovis infection through a dual mechanism-promoting autophagy and regulating cholesterol homeostasis-thereby reducing bacterial survival and protecting tissues from damage. This discovery provides a novel strategy for prevention and treatment of M. bovis infection, warranting further investigation in bovine models to assess pharmacokinetics, dosage, and clinical efficacy.