Dogs cast NETs too: Canine neutrophil extracellular traps in health and immune-mediated hemolytic anemia

•Dog neutrophils release NETs.•Nucleosomesmay be increased in IMHA.•Hemolysis or icterus falsely elevate nucleosomes in a commercial ELISA.

Neutrophil extracellular traps (NETs) are webs of DNA and protein with both anti-microbial and pro-thrombotic properties which have not been previously reported in dogs. To confirm dog neutrophils can form NETs, neutrophils were isolated from healthy dogs, and stimulated in vitro with 2 μM, 8 μM, 31 μM, and 125 μM platelet activating factor (PAF) or 0.03 μM, 0.1 μM, 0.4 μM, 1.6 μM and 6.4 μM phorbol-12-myristate-13-acetate (PMA). Extracellular DNA was measured using the cell impermeable dye Sytox Green every hour for 4 h. At 4 h, extracellular DNA was significantly greater than non-stimulated cells at concentrations ≥31 μM and ≥0.1 μM for PAF and PMA, respectively. Cells stimulated with 31.25 μM PAF reached maximal fluorescence by 1 h, whereas maximal fluorescence was not achieved until 2 h for cells stimulated with 0.1 μM PMA. Immunofluorescent imaging using DAPI and anti-elastase antibody confirmed that extracellular DNA is released as NETs.As NETs have been implicated in thrombosis, nucleosomes, a marker correlated with NET formation, were measured in the serum of dogs with the thrombotic disorder primary immune-mediated hemolytic anemia (IMHA) (n = 7) and healthy controls (n = 20) using a commercially available ELISA. NETs were significantly higher in IMHA cases than controls (median 0.12 and 0.90, respectively, p = 0.01), but there were large positive interferences associated with hemolysis and icterus.In summary, the study is the first to describe NET generation by canine neutrophils and provides preliminary evidence that a marker associated with NETs is elevated in IMHA. However, this apparent elevation must be interpreted with caution due to the effect of interference, emphasizing the need for a more specific and robust assay for NETs in clinical samples.