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

Figure 4

Participation of TLR2 in the internalization of S. aureus by bMECs inhibited with E2. (a) Primary cultures of bMECs treated for 24 h with E2 (50 pg/mL) were incubated with 5 μg/mL of a specific blocking antibody against TLR2 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 TLR2 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 TLR2 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 TLR2 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-TLR2 antibody overnight and analyzed with flow cytometry. Different letters indicate significant changes among treatments (). Vehicle: 1% ethanol.

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