Catching NETs—development of a flow cytometric assay to detect canine neutrophil extracellular traps
Date
2022-05
Authors
Larsen, Kristina Rhea
Major Professor
Advisor
Viall, Austin K
Bellaire, Bryan H
LeVine, Dana N
Miller, Cathy L
Committee Member
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Abstract
Neutrophil extracellular traps (NETs) contribute to the pathophysiology of various diseases via host tissue damage, hyperinflammation, and thrombosis across multiple species. Immune-mediated hemolytic anemia (IMHA), an autoimmune disorder of dogs, has a high mortality rate due to thrombotic events, but the exact role NETs play in the pathophysiology is still unclear and requires further investigation. It has been hypothesized that NETs, through their prothrombotic and proinflammatory properties, contribute to mortality in canine IMHA. However, a readily accessible specific test for canine NETs is lacking, thus exploring the role of NETs in IMHA pathogenesis has been limited. The most specific assay used to study NETosis is time- and labor-intensive immunofluorescence microscopy, which can be subjective and biased due to human error. Other assays, such as ELISAs, measure markers of NETs but are not specific for NETs. Flow cytometry is a quick and more objective method to specifically identify and quantitate NETs. There have been several protocols developed for use in humans and mice, using a variety of stains and gating schemes to identify NETs, but no such protocol has been developed for use in companion animals such as dogs. We have developed a flow-cytometry based assay to detect canine NETs using the DNA stain SYTOX Green, an anti-citrullinated H3 histone antibody, and an antibody for a neutrophil marker, CD11b. In isolated canine neutrophils, we were able to demonstrate that we could detect neutrophils using CD11b, we could detect PMA-stimulated increases in citH3 histone and extracellular DNA positive CD11b events, and we could detect significantly increased SYTOX-citH3-CD11b (+) events in PMA-stimulated samples versus the non-stimulated samples.
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