NETosis and Calcium influx in Dromedary Camel Neutrophils after in vitro Toll-like Receptor Stimulation

Abstract

Neutrophilic granulocytes are vital immune cells of the early response to pathogens. They contribute to the antimicrobial response through phagocytosis, production of reactive oxygen species, cytokine production, degranulation, and NET-formation. Neutrophil extracellular traps (NETs), also known as NETosis, are a critical antibacterial effector mechanism of cells of myeloid effector cells, including neutrophils and macrophages. Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) that mediate pathogen sensing through the recognition of microbial structures known as pathogen-associated molecular patterns (PAMPs). The present study aimed to investigate the potential of several TLR ligands that mimic the sensing of bacterial and viral pathogens to stimulate NET-formation or Ca2+ influx in camel neutrophils. Neutrophils were purified from blood and were stimulated in vitro with ligands to TLR4, TLR2/1, TLR7/8, or TLR3. Net-formation was analyzed using the DNA-sensitive dye SYTOX™ Green and staining with antibodies to the neutrophil's granular enzyme myeloperoxidase. Real-time stimulation-induced Ca2+ influx was measured using the Ca2+-sensitive dye Flou-4 and flow cytometry. Only the TLR4-ligand lipopolysaccharide (LPS) could induce NET-formation in camel neutrophils, while none of the investigated TLR agonists showed a Ca2+ influx-inducing effect in camel neutrophils. The current study represents the first report on the impact of direct activation of TLR on NET-formation and Ca2+ influx in camel neutrophils with a selective effect of LPS on NET-formation induction. Future studies may investigate the molecular mechanisms behind the different responsiveness of bovine and camel neutrophils to TLR stimulation.