PhD Student North Carolina State University Wendell, North Carolina, United States
Abstract:
Background: While neutrophil extracellular traps (NETs) are an important host response, dysregulated NET release (NETosis) causes off-target damage, perpetuating systemic inflammatory response syndrome (SIRS). The neutrophil-platelet interaction is upregulated during SIRS increasing NETosis. In SIRS, inflammatory cytokines and metabolic parameters are increased. Despite high mortality rates associated with SIRS in horses, methods to investigate NETs in clinically relevant in vitro models are limited.
Hypothesis/
Objectives: To determine if cytokines, lactate, and glucose, parameters elevated in SIRS, can model SIRS-associated NETosis in the presence or absence platelets.
Animals: Whole blood was obtained from 10 healthy, university owned horses.
Methods: Neutrophils alone or with platelets were primed with lipopolysaccharide, which does not reliably induce in vitro NETosis by equine neutrophils. TNF-alpha, IL-1beta, IL-8, GM-CSF, lactate (L- and D- isomers), and glucose were used as potential stimuli, phorbol 12-myristate 13-acetate (PMA) as the positive control, and unstimulated cells as the negative control. NETosis was measured using SYTOX Green nucleic acid stain over 6 hours, with confocal microscopy performed using NET specific antibodies (citrullinated histone H3 (CitH3) and myeloperoxidase).
Results: TNF-alpha significantly increased NET release in a concentration-dependent manner, both with and without platelets. Lactate isomers alone and in combination with glucose significantly promoted NETosis when neutrophils were co-incubated with platelets. CitH3 was upregulated under lactate isomer with glucose stimulated conditions. Myeloperoxidase was increased in the presence of platelets.
Conclusions and Clinical Importance: This study shows the complex nature of neutrophil-associated SIRS pathophysiology and a potential physiologic model to induce in vitro NETosis in horses.
