Effect of the synthetic Toll-like receptor ligands LPS, Pam3CSK4, HKLM and FSL-1 in the function of bovine polymorphonuclear neutrophils

• TLR ligands induce morphology changes and the phagocytosis activity in bovine PMN.
• Bovine PMN express TLR2 and TLR4 at the membrane surface.
• Pam3CSK4 induces calcium influx, ROS production and MMP-9 secretion.
• The ROS production induced by Pam3CSK4 was blocked by the SOCE inhibitor 2-APB.
• Incubation with TLR ligands does not affect the apoptosis of bovine PMN.

Toll-like receptors (TLR) are a family of pattern recognition receptors that sense microbial associated molecular patterns (MAMP) such as microbial membrane components and nucleic acids of bacterial origin. Polymorphonuclear neutrophils (PMN) are the first cell of the innate immune system to arrive at the site of infection or injury and elicit oxidative and non-oxidative microbicidal mechanisms. Observations in human and mouse suggest that TLR ligands can induce direct responses in PMN. So far, there is no information of the effect of synthetic TLR ligands on the response of bovine PMN. The objective of this study was to evaluate the functional response of bovine PMN incubated with four synthetic TLR ligands: ultrapure LPS (TLR4), Pam3CSK4 (TLR2/1), HKLM (TLR2) and FSL-1 (TLR2/6). The results show that all the ligands increment cells size as identified by changes in the FSC-SSC as part of the flow cytometric analysis. Interestingly, only Pam3CSK4 consistently induced a calcium influx, increased ROS production and secretion of gelatinase granules, whereas no response was seen using other ligands. Furthermore, exposure of bovine PMN to ultrapure LPS, Pam3CSK4, HKLM or FSL-1 for 24 hours did not impact on apoptosis of these cells. Our data provide evidence for a selective response of bovine PMNs to TLR ligands.