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

Highlights

• Dog neutrophils release NETs.

• Nucleosomesmay be increased in IMHA.

• Hemolysis or icterus falsely elevate nucleosomes in a commercial ELISA.

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 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.

Introduction

A wide range of infectious and inflammatory stimuli induce neutrophils to release webs of histone-associated DNA decorated with granular proteins (Remijsen et al., 2011). Pathogens become entangled in these neutrophil extracellular traps (NETs), where they are exposed to high concentrations of anti-microbial proteins such as bactericidal permeability increasing protein, neutrophil elastase, and myeloperoxidase (Urban et al., 2006). NETs are therefore important in controlling infection, but there is also growing evidence that they contribute to inflammatory tissue damage and thrombosis (Brinkmann et al., 2004, Knight and Kaplan, 2012, Martinod and Wagner, 2014).

NETs promote inflammatory tissue damage and thus contribute to pathology in a wide range of disorders. For example, NET components are important sources of autoantigens in autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis (Grayson and Kaplan, 2015). NETs also drive inflammation in these, and other diseases such as diabetes (Diana et al., 2013), sepsis (Luo et al., 2014) and transfusion related lung injury (Thomas et al., 2012), by mechanisms including complement activation (Wang et al., 2015), upregulation of IFNα (Diana et al., 2013) and inflammasome activation (Kahlenberg et al., 2013).

Neutrophil extracellular trap formation has not been previously investigated in dogs. Extracellular trap formation has been demonstrated in species ranging from invertebrates to humans, and it may seem safe to assume that dogs also form NETs (Robb et al., 2014, Remijsen et al., 2011). However, there are important species differences in the nature of NET formation, which limit the extent to which findings in one species can be extrapolated to another. For example, it has been recently demonstrated that myeloperoxidase inhibition greatly reduces NET formation in humans but has little impact in mice (Akong-Moore et al., 2012).

Future investigation of NET formation may have particular relevance to treatment of canine primary immune-mediated hemolytic anemia (IMHA). IMHA carries a grave prognosis, with mortality as high as 83% (Weinkle et al., 2005). Although the presenting signs of the disease are typically those of anemia due to autoantibody or complement mediated destruction of erythrocytes, the majority of these deaths are the result of thrombosis (Carr et al., 2002, Kidd and Mackman, 2013). Current understanding of thrombosis pathogenesis in IMHA is limited, and thromboprophylactic strategies are inadequate.

Conditions in IMHA are ideal for NET formation. Heme and hypoxia, both of which are generated in IMHA, are implicated in induction of NETs (Chen et al., 2014, McInturff et al., 2012; Martinod and Wagner, 2014). There is strong in vitro and in vivo evidence that NETs play multiple roles in induction, propagation and stabilization of thrombi (Martinod and Wagner, 2014; von Brühl et al., 2012). Elevations in markers correlated with NET formation, such as circulating DNA-histone complexes termed nucleosomes, are present in human patients with thrombotic diseases like thrombotic microangiopathies and ischemic stroke (Fuchs et al., 2012, Geiger et al., 2007). Most excitingly, murine models of stroke and deep vein thrombosis suggest DNase therapy to breakdown NETs has potential as a therapy to prevent thrombosis or reduce thrombosis induced tissue damage (De Meyer et al., 2012, von Brühl et al., 2012).

Given that IMHA conditions can fuel NET formation and the established role of NETs in thrombosis, we hypothesized that nucleosomes, a marker associated with but not specific for NETs will be elevated in dogs with spontaneous IMHA. Specifically, this study aimed to (1) establish effective concentrations of two agonists for canine NET induction in vitro; (2) compare the kinetics of NET formation for the two agonists and (3) determine if serum nucleosomes were higher in dogs with primary IMHA compared to healthy controls.