Anti-Inflammatory and Antimicrobial Effects of Estradiol in Bovine Mammary Epithelial Cells during Staphylococcus aureus Internalization

Figure 3

Role of α5β1 integrin during the internalization of S. aureus into bMECs inhibited with E2. (a) Primary cultures of bMECs treated for 24 h with E2 (50 pg/mL) were incubated with 10 μg/mL of a specific blocking antibody against α5β1 integrin for 1 h and then challenged with S. aureus (MOI 30 : 1 bacteria per cell). The number of internalized bacteria is represented by the ratio of CFU/bMEC recovered after lysis of bMECs. (b) The α5β1 mRNA expression was analyzed by RT-qPCR and GAPDH was used as endogenous gene in all conditions. Fold-change values greater than 2 or less than 0.5 were considered as significant differentially expressed mRNAs. Each bar shows the mean of triplicates ± SD of three independent experiments. (c) The relative fluorescence intensities of α5β1 membrane abundance in bMECs treated with E2 and infected with S. aureus are shown. Fluorescence intensity was estimated from 10,000 events. An inserted histogram plot that shows α5β1 staining data in vehicle-treated bMECs (black line), cells that were challenged with S. aureus (blue line), cells that were stimulated with E2 (red line), and E2-treated bMECs infected with S. aureus (green line). The cells were fixed and stained extracellularly with an anti-α5β1 antibody overnight and analyzed by flow cytometry. (d) The abundance of α5β1 integrin in the bMEC membrane determined as the mean of the fluorescence of challenged bMECs at different times of infection with the same MOI (30 : 1 bacteria per cell). A total of 10,000 events were measured. Different letters indicate significant changes among treatments (), except for (b), where different letters above the bars indicate significant changes among the four treatments within the same gene evaluated (). Vehicle: 1% ethanol.

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