Equine neutrophils selectively release neutrophil extracellular traps (NETs) in response to chemical and bacterial agonists

Breanna Sheahan ^1,2* Alicia G Schubert ^1,2 William Schubert ³ M. Katie Sheats ^1,2 Lauren Virginia Schnabel ^1,2 Jessica M Gilbertie ^1,2,4*

• ¹ Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina, United States
• ² Comparative Medicine Institute, North Carolina State University, Raleigh, North Carolina, United States
• ³ College of William & Mary, Williamsburg, Virginia, United States
• ⁴ Department of Biomedical Sciences and Pathobiology, Virginia Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, Virginia, United States

Neutrophil extracellular traps (NETs) likely play a significant role in response to a variety of infectious and inflammatory stimuli in human and veterinary medicine. Although entrapment of bacteria can be an important function of NETs, the exuberant release of DNA and other intracellular molecules has also been implicated in the pathogenesis of different diseases. Thus, the process of NET formation must be tightly controlled and represents a therapeutic opportunity for interventions. Horses are particularly sensitive to bacterial stimuli that have previously been shown to cause NETs in other species, but the species-specific processes that control NET release have not been fully elucidated. The purpose of this study was to compare the magnitude of response of equine neutrophils to different chemical and bacterial stimuli, including phorbol 12-myristate 13-acetate (PMA), a calcium ionophore (A23187), Staphylococcus aureus, and Escherichia coli. In addition, we investigated whether ex vivo equine NET formation is controlled by the NADPH-oxidase (NOX) pathway and by autophagy, both of which control NET formation in other species. We demonstrated that equine neutrophils produce robust NETs in response to calcium ionophore and E. coli stimuli and produce fewer NETs in response to PMA and S. aureus. Both NOX-dependent and NOXindependent pathways of NET formation were identified in equine neutrophils. Autophagy inhibition altered the mechanics of NET release, appearing to reduce the amount of extracellular DNA stranding. These results should provide insight into equine-specific neutrophil biology, which could be key for managing equine diseases such as asthma and laminitis.