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

Figure 8

Expression and production of antimicrobial molecules in bMECs treated with E2 and infected with S. aureus. (a) S. aureus viability from supernatants of infection assays performed in the absence of gentamicin. Noninternalized bacteria viability was analyzed by flow cytometry using the LIVE/DEAD BacLight Bacterial viability kit. Fluorescence intensity was estimated from 10,000 events. Values were determined considering the effect of control (bMECs cultured with the vehicle 1% ethanol) as 100% of viability. (b) Effect of conditioned medium from bMECs treated with vehicle (1% ethanol) or E2 (50 pg/mL) for 24 h on S. aureus viability. Bacteria were incubated 2 h with conditioned media and viability was analyzed by flow cytometry using the LIVE/DEAD BacLight Bacterial viability kit. Fluorescence intensity was estimated from 10,000 events. Values were determined considering the effect of control-conditioned medium (bMECs cultured with the vehicle 1% ethanol) as 100% of viability. (c) RT-qPCR analysis showing the mRNA expression levels of DEFB1, BNBD4, BNBD5, BNBD10, LAP, TAP, and S100A7. The bMECs were treated with 50 pg/mL of E2 for 24 h and then challenged with S. aureus for 2 h. The data are presented as the ratio of the target gene expression compared with the expression level of GAPDH. Each bar shows the mean of triplicates ± SD of three independent experiments. Fold-change values greater than 2 or less than 0.5 were considered as significant differentially expressed mRNAs. (d) Concentration of DEFB1 in culture medium of bMECs treated with 50 pg/mL of E2 for 24 h and then challenged with S. aureus for 2 h. The protein concentrations were determined by ELISA. Each bar shows the mean of duplicates ± SD from different experiments, nd: nondetermined. (e) Nitric oxide production in bMECs treated with 50 pg/mL of E2 for 24 h and then challenged with S. aureus for 2 h, measured as NO₂ concentration in culture medium. Each bar shows the mean of triplicates ± SE of three independent experiments. Different letters indicate significant changes among treatments (), except for (c), where different letters above the bars indicate significant changes among the four treatments within the same gene evaluated (). Vehicle: 1% ethanol.

(a)

(b)

(c)

(d)

(e)