title: Dogs cast NETs too: Canine neutrophil extracellular traps in health and immune-mediated hemolytic anemia.
abstract: 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 2μM, 8μM, 31μM, and 125μM platelet activating factor (PAF) or 0.03μM, 0.1μM, 0.4μM, 1.6μM and 6.4μM phorbol-12-myristate-13-acetate (PMA). Extracellular DNA was measured using the cell impermeable dye Sytox Green every hour for 4h. At 4h, extracellular DNA was significantly greater than non-stimulated cells at concentrations ≥31μM and ≥0.1μM for PAF and PMA, respectively. Cells stimulated with 31.25μM PAF reached maximal fluorescence by 1h, whereas maximal fluorescence was not achieved until 2h for cells stimulated with 0.1μM 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.
authors.affiliation: Department of Veterinary Microbiology and Preventative Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USA. Electronic address: ublocke@iastate.edu.
authors.forename: Unity
authors.initials: U
authors.lastname: Jeffery
authors.affiliation: Department of Veterinary Microbiology and Preventative Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USA.
authors.forename: Kayoko
authors.initials: K
authors.lastname: Kimura
authors.affiliation: MRC Centre for Inflammation Research, The Queen's Medical Research Institute, University of Edinburgh, Edinburgh EH16 4TJ, Scotland, UK.
authors.forename: Robert
authors.initials: R
authors.lastname: Gray
authors.affiliation: Department of Veterinary Microbiology and Preventative Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USA.
authors.forename: Paul
authors.initials: P
authors.lastname: Lueth
authors.affiliation: Department of Veterinary Microbiology and Preventative Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USA.
authors.forename: Bryan
authors.initials: B
authors.lastname: Bellaire
authors.affiliation: Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USA.
authors.forename: Dana
authors.initials: D
authors.lastname: LeVine
mesh_terms: D000744:Anemia, Hemolytic, Autoimmune / Q000276:immunology / Q000378:metabolism / Q000662:veterinary*; D000818:Animals; D016022:Case-Control Studies; D016923:Cell Death; D004247:DNA; D004283:Dog Diseases / Q000276:immunology / Q000378:metabolism*; D004285:Dogs; D065206:Extracellular Traps / Q000502:physiology*; D005260:Female; D008297:Male; D015203:Reproducibility of Results
chemical_list: D004247:DNA
keywords: Dog; Immune-mediated hemolytic anemia; NET; Neutrophil extracellular trap; Nucleosome