Potential involvement of NETs in the pathogenesis of SRMA.

Potential involvement of NETs in the pathogenesis of SRMA.</p

Correlation analysis: Association between H3Cit levels and DNase activity in serum and CSF and different parameters in SRMA acute group.

Correlation analysis: Association between H3Cit levels and DNase activity in serum and CSF and different parameters in SRMA acute group.</p

CSF and serum samples for H3Cit ELISA and DNase activity assay.

CSF and serum samples for H3Cit ELISA and DNase activity assay.</p

Median concentrations and ranges of H3Cit levels (ng/mL) in CSF and serum of different disease groups.

Median concentrations and ranges of H3Cit levels (ng/mL) in CSF and serum of different disease groups.</p

Spiky and cloudy NETs in a 7-month-old female Beagle.

(A) Neutrophil extracellular traps (NET) as combined extracellular DNA-fibers presented as cloudy NET-formations with staining of NET-specific citrullinated histone H3 (H3Cit) and DNA-histone-1-complexes were detected in the cerebrospinal fluid during acute onset. Blue = counterstaining of DNA (Hoechst), green = DNA/histone-1-complexes (NETs), red = citrullinated histone H3 (H3Cit). Representative images are shown. Scale bar = 100 μm. (B), (C) Spiky NET-formations were detected. Blue = DNA (Hoechst), green = DNA/histone-1-complexes (NETs), red = citrullinated histone (H3Cit). Scale bar = 20 μm. (D) Settings of the immunofluorescence images were adjusted to a respective isotype control. Respective images of the respective isotype control are presented. Scale bar = 100 μm.</p

Correlation analysis: Association between H3Cit levels and DNase activity in serum and CSF in SRMA acute group.

Correlation analysis: Association between H3Cit levels and DNase activity in serum and CSF in SRMA acute group.</p

Results H3Cit ELISA and DNase activity assay.

In steroid-responsive meningitis-arteritis (SRMA), inflammatory dysregulation is driven by neutrophilic granulocytes resulting in purulent leptomeningitis. Neutrophils can generate neutrophil extracellular traps (NET). Uncontrolled NET-formation or impaired NET-clearance evidently cause tissue and organ damage resulting in immune-mediated diseases. The aim of the study was to verify that NET-formation is detectable in ex vivo samples of acute diseased dogs with SRMA by visualizing and measuring NET-markers in serum and cerebrospinal fluid (CSF) samples. CSF-samples of dogs with acute SRMA (n = 5) and in remission (n = 4) were examined using immunofluorescence (IF)-staining of DNA-histone-1-complexes, myeloperoxidase and citrullinated Histone H3 (H3Cit). Immunogold-labeling of H3Cit and neutrophil elastase followed by transmission electron microscopy (TEM) were used to determine ultrastructural NET-formation in the CSF of one exemplary dog. H3Cit-levels and DNase-activity were measured in CSF and serum samples using an H3Cit-ELISA and a DNase-activity-assay, respectively in patients with the following diseases: acute SRMA (n = 34), SRMA in remission (n = 4), bacterial encephalitis (n = 3), meningioma with neutrophilic inflammation (n = 4), healthy dogs (n = 6). NET-formation was detectable with IF-staining in n = 3/5 CSF samples of dogs with acute SRMA but were not detectable during remission. Vesicular NET-formation was detectable in one exemplary dog using TEM. DNase-activity was significantly reduced in dogs suffering from acute SRMA compared to healthy control group (p </div

Ultrastructural NET-Formation of an 11-month-old male Labrador Retriever.

Representative image (3A) of CSF neutrophils and the respective magnifications (3B) of extracellular NET-Formation in this individual patient were shown in both images. Citrullinated histone H3 (H3Cit) (white arrowhead) and neutrophil elastase (NE) (white arrow) were detected with immunogold-labeling in neutrophil granulocytes taken from CSF samples during acute onset of SRMA (gold/H3Cit = 5 nm, gold/neutrophil elastase = 10 nm). Examination was performed via transmission electron microscopy. Black asterisks show filamentous chromatin decorated with H3Cit and NE representing NET structures (3B). NET-components like H3Cit and NE were budding near the cell membrane forming spiky convexities at the cell membrane and show the release of NETs in the extracellular space (black arrowheads) (3B). Left image (3A): scale bar = 2 μm. Right image (3B): scale bar = 500 nm.</p

11-month-old male Labrador Retriever with SRMA.

Representative images (A1, B1) of CSF neutrophils and respective magnifications of ultrastructural processes of intracellular NET-Formation in this individual patient were shown in each row. Citrullinated histone H3 (H3Cit) and neutrophil elastase (NE) were detected with immunogold-labeling in neutrophil granulocytes taken from CSF samples during acute onset of SRMA (gold/H3Cit = 5 nm, gold/neutrophil elastase = 10 nm). Examination was performed via transmission electron microscopy. NE (white arrows) was present in the nucleus as definite indicator for early NETosis and catalyst of chromatin decondensation in cooperation with myeloperoxidase (MPO) (A2, B2). Colocalized H3Cit and NE were present with high amounts in multiple, nuclear, light grey vesicles (black arrow) in the cytoplasm in A2, A3, B2, B3 representing intracellular created NETs. NE was present in dark grey neutrophil granules (white arrowheads) (A2, A3, B2, B3). Left column: scale bar = 5 μm. Middle and right column: scale bar = 500 nm. (TIF)</p

Correlation analysis: Association between H3Cit levels and DNase activity in CSF and serum and different parameters in SRMA acute.

Correlation analysis: Association between H3Cit levels and DNase activity in CSF and serum and different parameters in SRMA acute.</p