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 tarsus 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. mew internalization into bMECs was reduced in cells pre-treated with Thi2.1 at 5 and 10 mu g/mL during 24 h, effect related to the participation of TLR2. In addition, bMECs pre-treated with Thi2.1 (24 h) significantly increased TNF-alpha (similar to 2-fold) and IL-6 (similar to 7-fold), whereas decreased IL-10 gene expression (similar to 0.5-fold). Interestingly, Thi2.1 inhibits the up-regulation induced by S. mucus of TNF-alpha and IL-10 gene expression, as well as NO production. In addition, Thi2.1 (10 mu g/mL) up-regulates the expression of the chemokine IL-8 (similar to 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. caucus internalization. Some of these effects are mediated by TLR2.