Staphylococcal enterotoxin M induced inflammation and impairment of bovine mammary epithelial cells

摘要

Staphylococcus aureus is one of the major etiologicalpathogens of bovine mastitis. Its invasion into mammaryepithelial cells has been proven to be a key eventin the pathogenesis of mastitis. However, the specificpathogenic factors have not been clearly identified.Staphylococcus aureus often triggers infections by releasingvirulence factor. Recent several studies reportedthat staphylococcal enterotoxin M was one of the mostfrequently found enterotoxin genes associated withbovine mastitis. Thus, the effect of staphylococcal enterotoxinM on inflammation and damage of the bovinemammary epithelial bovine mammary gland epithelialcell line (MAC-T) cells with 48 h treatment was exploredin the present study. First, staphylococcal enterotoxinM protein was purified by a Ni-NTA spin column (GELife Science, Westborough, MA). The levels of tumornecrosis factor-α, IL-6, and monocyte chemoattractantprotein 1 (MCP-1) secretion were measured with thecorresponding ELISA kits (R&D Systems, Abingdon,UK). Second, cell viability was assessed with a CellCounting Kit-8 (Bioswamp, Wuhan, China) and theapoptotic percentage of cells was determined by annexinV-fluorescein isothiocyanate (FITC)/propidiumiodide (PI; Beyotime, Nanjing, China) staining. Third,ATP concentration, reactive oxygen species (ROS)generation and lactate dehydrogenase (LDH) releasewere assayed with commercial kits, then mitochondrialmembrane potential (ΔΨm) was estimated usingfluorescent probe JC-1 (Beyotime). Finally, the productionintercellular cell adhesion molecule-1 (ICAM-1),microtubule-associated protein 1A/1B-light chain 3 I/II (LC3 I/II), p62 (Proteintech, Rosemont, IL), andphosphorylation of IκBα, caspase 3, and mammaliantarget of rapamycin were detected by Western blot.The results showed that staphylococcal enterotoxin Minduced inflammation of epithelial cells (upregulatingtumor necrosis factor-α, IL-6, MCP-1, and ICAM-1production) and activated NF-κB (promoting phosphorylationof IκBα). Furthermore, staphylococcalenterotoxin M impaired MAC-T cells via cell necrosis(enhancing LDH release), apoptosis (annexin V-FITC/PI stain, exacerbating oxidative stress, decreasingΔΨm and intracellular ATP concentration, and activatingcaspase 3), but independent of autophagy (nonsignificantlyincreasing LC3-II, decreasing p62 expression,and activating mammalian target of rapamycin).Thereby, staphylococcal enterotoxin M induced theinflammatory property of bovine mammary epithelialcells by boosting cytokine, chemokine, and adhesionmolecule production. Furthermore, it caused epithelialcell dysfunction via depressing cell viability and initiatingcell necrosis and apoptosis. Because epithelial cellsplayed important roles in orchestrating the inflammatoryresponse and protecting bovine mammary tissuefrom mastitis, our results indicated that staphylococcalenterotoxin M may be associated with mastitis.