Immunomodulatory effects of thionin Thi2.1 from Arabidopsis thaliana on bovine mammary epithelial cells.

Antimicrobial peptides (AMPs) are key elements of plant defense mechanisms, resembling conserved protection strategies also present in mammals. Among the AMPs, plant thionins are particularly interesting due that display antibacterial and antifungal activities. In Arabidopsis thaliana have been described four thionins: Thi2.1, Thi2.2, Thi2.3 and Thi2.4. Work from our group shows that Thi2.1 expressed by bovine endothelial cells has direct antibacterial activity against Staphylococcus aureus mastitis isolates, bacteria able to persist inside bovine mammary epithelial cells (bMECs). Thus, the objective of this work was to analyze the immunomodulatory effects of the AMP thionin Thi2.1 from A. thaliana on bMECs during S. aureus infection. According to the results, S. aureus internalization into bMECs was reduced in cells pre-treated with Thi2.1 at 5 and 10 μg/mL during 24 h, effect related to the participation of TLR2. In addition, bMECs pre-treated with Thi2.1 (24 h) significantly increased TNF-α (~2-fold) and IL-6 (~7-fold), whereas decreased IL-10 gene expression (~0.5-fold). Interestingly, Thi2.1 inhibits the up-regulation induced by S. aureus of TNF-α and IL-10 gene expression, as well as NO production. In addition, Thi2.1 (10 μg/mL) up-regulates the expression of the chemokine IL-8 (~3-fold) in infected bMECs. Some of these effects are related to TLR2 activation. In this sense, Thi2.1 also reduces S. aureus-induced TLR2 gene expression and membrane abundance. In conclusion, Thi2.1 from A. thaliana modulates bMEC innate immune response by inducing the production of pro- and anti-inflammatory molecules while inhibits S. aureus internalization. Some of these effects are mediated by TLR2.