RESUMO
Epigenetic mechanisms are essential in the regulation of immune response during infections. Changes in the levels of reproductive hormones, such as prolactin, compromise the mammary gland's innate immune response (IIR); however, its effect on epigenetic marks is poorly known. This work explored the epigenetic regulation induced by bovine prolactin (bPRL) on bovine mammary epithelial cells (bMECs) challenged with Staphylococcus aureus. In this work, bMECs were treated as follows: (1) control cells without any treatment, (2) bMECs treated with bPRL (5 ng/ml) at different times (12 or 24 h), (3) bMECs challenged with S. aureus for 2 h, and (4) bMECs treated with bPRL at different times (12 or 24 h), and then challenged with S. aureus 2 h. By western blot analyses of histones, we determined that the H3K9ac mark decreased (20%) in bMECs treated with bPRL (12 h) and challenged with S. aureus, while the H3K9me2 mark was increased (50%) in the same conditions. Also, this result coincided with an increase (2.3-fold) in HDAC activity analyzed using the cellular histone deacetylase fluorescent kit FLUOR DE LYS®. ChIP-qPCRs were performed to determine if the epigenetic marks detected in the histones correlate with enriched marks in the promoter regions of inflammatory genes associated with the S. aureus challenge. The H3K9ac mark was enriched in the promoter region of IL-1ß, IL-10, and BNBD10 genes (1.5, 2.5, 7.5-fold, respectively) in bMECs treated with bPRL, but in bMECs challenged with S. aureus it was reduced. Besides, the H3K9me2 mark was enriched in the promoter region of IL-1ß and IL-10 genes (3.5 and 2.5-fold, respectively) in bMECs challenged with S. aureus but was inhibited by bPRL. Additionally, the expression of several miRNAs was analyzed by qPCR. Let-7a-5p, miR-21a, miR-30b, miR-155, and miR-7863 miRNAs were up-regulated (2, 1.5, 10, 1.5, 3.9-fold, respectively) in bMECs challenged with S. aureus; however, bPRL induced a down-regulation in the expression of these miRNAs. In conclusion, bPRL induces epigenetic regulation on specific IIR elements, allowing S. aureus to persist and evade the host immune response.
RESUMO
Changes in the levels of reproductive hormones compromise the bovine innate immune response (IIR). Changes in 17ß-estradiol (E2) and prolactin (bPRL) levels affect the IIR of bovine mammary epithelial cells (bMECs), the target tissue of these hormones. In this work, we explored the effect of the combined hormones on bMEC IIR during Staphylococcus aureus infection, and if they can modulate epigenetic marks. By gentamicin protection assays, we determined that combined hormones (bPRL (5 ng/mL) and E2 (50 pg/mL)] decrease S. aureus internalization into bMECs (~50%), which was associated with a reduction in integrin α5ß1 membrane abundance (MA) (~80%) determined by flow cytometry. Additionally, combined hormones increased Toll-like receptor 2 (TLR2) MA (25%). By RT-qPCR, we showed that combined hormones induce the expression of pro- and anti-inflammatory cytokine genes, as well as up-regulate antimicrobial peptide gene expression. The combined hormones induced H3K9Ac at 12 h of treatment, which coincides with the reduction in histone deacetylase (HDAC, 15%) activity. In addition, hormones increased the H3K9me2 mark at 12 h, which correlates with a reduction in the expression of KDM4A. In conclusion, bPRL and E2 modulate the IIR of bMECs, an effect that can be related to the regulation of histone H3 modifications such as H3K9Ac and H3K9me2.
