Graduate Research Assistant North Carolina State University Raleigh, NC, United States
Abstract:
Background: Severe equine asthma (sEA) is a chronic, inflammatory lower-airway disease characterized by cough, increased respiratory rate and effort at rest, and elevated lower airway neutrophils. Neutrophils contribute to ongoing inflammation and tissue damage through the release of proteolytic enzymes, reactive oxygen species and neutrophil extracellular traps (NETs.) NETs are extruded strands of DNA intercalated with histones and enzymatic proteins. Given their role in inflammation and tissue damage, NETs represent a potential therapeutic target for sEA. Hypothesis: NETs are increased in the lower airways of horses with sEA and inhibition of MARCKS with BIO-11006 will attenuate NETosis in equine neutrophils. Animals: 52 university-owned teaching/research horses and client-owned horses.
Methods: Cell free DNA (cfDNA) concentration was measured in BAL supernatant using the Qubit 4 fluorometer. Immunocytochemistry stained for two additional components of NETs: myeloperoxidase, and citrullinated-histone 3. Peripheral blood neutrophils were isolated, pretreated with BIO-11006 (MARCKS inhibitor) or RNS (control) and stimulated. CfDNA was quantified with SYTOX green.
Results: CfDNA was significantly (p < 0.05) higher in BAL supernatant from horses with sEA compared to healthy horses and horses with non-neutrophilic asthma. Confocal microscopy revealed NETs in BAL cytospins from horses with sEA, but not in cytospins from healthy horses. BIO-11006 significantly decreased A23187-induced NET formation in peripheral blood neutrophils.
Conclusions: These findings support further investigation of NETs as a therapeutic target for sEA. Next steps include determining the effect of nebulized MARCKS-inhibitor peptides on in vivo NETs in horses with sEA.
