Figure 4.

Induction of Toll-like receptor (TLR)-dependent NF-κB activation by E. bovis sporozoites. In order to investigate the activation of TLRs in bovine PMN, we used HEK cells expressing either bovine TLR2 (A) or a combination of bovine TLR4/MD2 (B). Cells were exposed to different E. bovis sporozoite preparations: live, heat killed (HK) or antigen (EbAg) for 24 h and assessed for activation of transcription factor NF-κB using a luciferase reporter assay. (A) HK sporozoites and EbAg induced substantial TLR2-dependent NF-κB activation compared to media alone (*** p < 0.0001, ** p < 0.01 and * p < 0.05, respectively). TLR2-induced NF-κB significantly increases when exposed to EbAg compared to live E. bovis (p < 0.05). (B) HK sporozoites induced a significant NF-κB response when compared to media (p < 0.05) and when compared to live parasitic stages (p < 0.01). A significant increase in NF-κB response was observed in EbAg when compared to media (p < 0.05). In both experiments, Pam₃CSK₄ and Lipopolysaccharides (LPS) served as ligand controls for TLR2 and TLR4/MD2, respectively, and phorbol 12-myristate 13-acetate (PMA) was used as an NF-κB technical control (data not shown for clarity). Data are represented as the mean of 3 replicates ± SD and were analyzed using an unpaired Student’s t-test using GraphPad Prism V.8.4.3 (GraphPad Software Inc.).