Heme oxygenase activates calcium release from the endoplasmic reticulum of bovine mammary epithelial cells to promote TFEB entry into the nucleus to reduce the intracellular load of Mycoplasma bovis.

Heme oxygenase HO-1 (HMOX) regulates cellular inflammation and apoptosis, but its role in regulation of autophagy in Mycoplasma bovis infection is unknown. The objective was to determine how the HO-1/CO- Protein kinase RNA-like endoplasmic reticulum kinase (PERK)-Ca2+- transcription factor EB (TFEB) signaling axis induces autophagy and regulates clearance of M. bovis by bovine mammary epithelial cells (bMECs). M. bovis inhibited autophagy and lysosomal biogenesis in bMECs and suppressed HO-1 protein and expression of related proteins, namely nuclear factor erythroid 2-related factor 2 (Nrf2) and Kelch-like ECH-associated protein 1 (keap1). Activation of HO-1 and its production of carbon monoxide (CO) were required for induction of autophagy and clearance of intracellular M. bovis. Furthermore, when HO-1 was deficient, CO sustained cellular autophagy. HO-1 activation increased intracellular calcium (Ca2+) and cytosolic localization activity of TFEB via PERK. Knockdown of PERK or chelation of intracellular Ca2+ inhibited HO-1-induced M. bovis autophagy and clearance. M. bovis infection affected nuclear localization of lysosomal TFEB in the MiT/TFE transcription factor subfamily, whereas activation of HO-1 mediated dephosphorylation and intranuclear localization of TFEB, promoting autophagy, lysosomal biogenesis and autophagic clearance of M. bovis. Nuclear translocation of TFEB in HO-1 was critical to induce M. bovis transport and survival of infected bMECs. Furthermore, the HO-1/CO-PERK-Ca2+-TFEB signaling axis induced autophagy and M. bovis clearance, providing a viable approach to treat persistent M. bovis infections.

Antioxidative Sirt1 and the Keap1-Nrf2 Signaling Pathway Impair Inflammation and Positively Regulate Autophagy in Murine Mammary Epithelial Cells or Mammary Glands Infected with Streptococcus uberis.

Streptococcus uberis mastitis in cattle infects mammary epithelial cells. Although oxidative responses often remove intracellular microbes, S. uberis survives, but the mechanisms are not well understood. Herein, we aimed to elucidate antioxidative mechanisms during pathogenesis of S. uberis after isolation from clinical bovine mastitis milk samples. S. uberis's in vitro pathomorphology, oxidative stress biological activities, transcription of antioxidative factors, inflammatory response cytokines, autophagosome and autophagy functions were evaluated, and in vivo S. uberis was injected into the fourth mammary gland nipple of each mouse to assess the infectiousness of S. uberis potential molecular mechanisms. The results showed that infection with S. uberis induced early oxidative stress and increased reactive oxygen species (ROS). However, over time, ROS concentrations decreased due to increased antioxidative activity, including total superoxide dismutase (T-SOD) and malondialdehyde (MDA) enzymes, plus transcription of antioxidative factors (Sirt1, Keap1, Nrf2, HO-1). Treatment with a ROS scavenger (N-acetyl cysteine, NAC) before infection with S. uberis reduced antioxidative responses and the inflammatory response, including the cytokines IL-6 and TNF-α, and the formation of the Atg5-LC3II/LC3I autophagosome. Synthesis of antioxidants determined autophagy functions, with Sirt1/Nrf2 activating autophagy in the presence of S. uberis. This study demonstrated the evasive mechanisms of S. uberis in mastitis, including suppressing inflammatory and ROS defenses by stimulating antioxidative pathways.

Mycoplasma bovis-generated reactive oxygen species and induced apoptosis in bovine mammary epithelial cell cultures.

Mycoplasma bovis is an important cause of bovine mastitis in China and worldwide. We hypothesized that M. bovis damages bovine mammary epithelial cells (bMEC), with the degree of damage varying among field isolates. Our objective was to evaluate 2 novel sequence type (ST) field strains of M. bovis (ST172 and ST173) for their ability to induce oxidative stress, cytotoxicity, pathomorphological changes, and apoptosis in bMEC, as a model for pathogenesis of M. bovis-induced bovine mastitis. Cytotoxicity (as indicated by release of lactate dehydrogenase, LDH) from bMEC depended on multiplicity of infection (MOI), with a high MOI (1:1,000) being required to induce cytotoxicity. Morphological changes in bMEC, including shrinkage, loss of cell integrity, and heavy staining (hematoxylin and eosin) of cytoplasm were apparent 24 h after infection with ST172 or ST173 M. bovis, with more severe changes being induced by the latter strain. Adhesion and invasion assays both had curvilinear patterns, peaking 12 h after infection with MOI of 1:1,000. Both production of reactive oxygen species (ROS) and proportion of apoptotic cells increased with time after infection. Increased Bax/Bcl-2 ratios and activation of caspase-3 implied involvement of mitochondria-dependent pathways of apoptosis. Furthermore, intracellular ROS generation, apoptosis, and cleaved caspase-3 were mitigated by N-acetyl-l-cysteine, a ROS scavenger. Both interleukin (IL)-1ß and IL-6 were significantly upregulated by ST172 and ST173 M. bovis, with little change in expression of tumor necrosis factor-α. One ST173 M. bovis isolate had the greatest cytotoxicity of all of our field isolates, with the highest LDH release, adhesion, invasion, ROS production, and apoptosis. In conclusion, our hypothesis was supported: M. bovis damaged bMEC by generating ROS and initiating a mitochondria-dependent pathway of apoptosis, with the degree of damage varying among field isolates. This study provided new knowledge regarding pathogenesis of M. bovis-induced bovine mastitis.