Equine Surgery Resident and PhD Candidate Louisiana State University School of Veterinary Medicine Baton Rouge, LA, United States
Abstract:
Background: IL-8 has been reported to induce Neutrophil Extracellular Trap formation in people, but its effect on equine neutrophils is unknown. Activation of NETs can occur through NOX2-dependent or PAD4-dependent pathways; however, specific activation of these pathways in the horse is not well understood. Hypothesis/
Objectives: IL-8 will induce NETosis through the PAD4-dependent pathway in neutrophils isolated from healthy horses. Animals: Four healthy horses from the university research herd.
Methods: Isolated neutrophils were stimulated with known NET inducers (A213817, PMA, reIL-8). NET formation was assessed through live-cell imaging with cytotox green dye, western hybridization for citrullination of histone H3, and immunofluorescence of MPO/DAPI. For live cell analysis, number of NETs formed was determined for 7 hours, and data were analyzed using two-way repeated measure ANOVA with Tukey’s post hoc comparison between groups. Band intensities and microscopic formation of NETs were subjectively evaluated and compared between groups.
Results: Stimulation with PMA resulted in NET formation (p < 0.05) starting at 4 hours compared to reIL-8 and media control groups when evaluated through live cell imaging. Strong band intensity was observed in neutrophils stimulated with A23817 for 6 hours compared to PMA and media control. reIL-8 stimulation induced NETosis in a concentration-dependent manner, with mild increases in band intensity compared to PMA and media control. Conclusions and Clinical Importance: While both pathways of NETosis can be induced in healthy equine neutrophils, IL-8 appears to mildly stimulate NETosis through the PAD4-dependent pathway. NETosis in clinical cases and assessment of NET inhibitors remain to be assessed.
