Hexadecanamide alleviates Staphylococcus aureus-induced mastitis in mice by inhibiting inflammatory responses and restoring blood-milk barrier integrity.

Subacute ruminal acidosis (SARA) has been demonstrated to promote the development of mastitis, one of the most serious diseases in dairy farming worldwide, but the underlying mechanism is unclear. Using untargeted metabolomics, we found hexadecanamide (HEX) was significantly reduced in rumen fluid and milk from cows with SARA-associated mastitis. Herein, we aimed to assess the protective role of HEX in Staphylococcus aureus (S. aureus)- and SARA-induced mastitis and the underlying mechanism. We showed that HEX ameliorated S. aureus-induced mastitis in mice, which was related to the suppression of mammary inflammatory responses and repair of the blood-milk barrier. In vitro, HEX depressed S. aureus-induced activation of the NF-κB pathway and improved barrier integrity in mouse mammary epithelial cells (MMECs). In detail, HEX activated PPARα, which upregulated SIRT1 and subsequently inhibited NF-κB activation and inflammatory responses. In addition, ruminal microbiota transplantation from SARA cows (S-RMT) caused mastitis and aggravated S. aureus-induced mastitis, while these changes were reversed by HEX. Our findings indicate that HEX effectively attenuates S. aureus- and SARA-induced mastitis by limiting inflammation and repairing barrier integrity, ultimately highlighting the important role of host or microbiota metabolism in the pathogenesis of mastitis and providing a potential strategy for mastitis prevention.

Ferroptosis is involved in Staphylococcus aureus-induced mastitis through autophagy activation by endoplasmic reticulum stress.

Cell death caused by severe Staphylococcus aureus (S. aureus) infection is a fatal threat to humans and animals. However, whether ferroptosis, an iron-dependent form of cell death, is involved in S. aureus-induced cell death and its role in S. aureus-induced diseases are unclear. Using a mouse mastitis model and mammary epithelial cells (MMECs), we investigated the role of ferroptosis in the pathogenesis of S. aureus infection. The results revealed that S. aureus-induced ferroptosis in vivo and in vitro as demonstrated by dose-dependent increases in cell death; the level of malondialdehyde (MDA), the final product of lipid peroxidation; and dose-dependent decrease the production of the antioxidant glutathione (GSH). Treatment with typical inhibitors of ferroptosis, including ferrostatin-1 (Fer-1) and deferiprone (DFO), significantly inhibited S. aureus-induced death in MMECs. Mechanistically, treatment with S. aureus activated the protein kinase RNA-like ER kinase (PERK)-eukaryotic initiation factor 2, α subunit (eIF2α)-activating transcription factor 4 (ATF4)-C/EBP homologous protein (CHOP) pathway, which subsequently upregulated autophagy and promoted S. aureus-induced ferroptosis. The activation of autophagy degraded ferritin, resulting in iron dysregulation and ferroptosis. In addition, we found that excessive reactive oxygen species (ROS) production induced ferroptosis and activated endoplasmic reticulum (ER) stress, manifesting as elevated p-PERK-p-eIF2α-ATF4-CHOP pathway protein levels. Collectively, our findings indicate that ferroptosis is involved in S. aureus-induced mastitis via ER stress-mediated autophagy activation, implying a potential strategy for the prevention of S. aureus-associated diseases by targeting ferroptosis. In conclusion, the ROS-ER stress-autophagy axis is involved in regulating S. aureus-induced ferroptosis in MMECs. These findings not only provide a new potential mechanism for mastitis induced by S. aureus but also provide a basis for the treatment of other ferroptotic-related diseases.