Methods for the Assessment of NET Formation: From Neutrophil Biology to Translational Research

Abstract

Several studies have indicated that a neutrophil extracellular trap (NET) formation, apart from its role in host defense, can contribute to or drive pathogenesis in a wide range of inflammatory and thrombotic disorders. Therefore, NETs may serve as a therapeutic target or/and a diagnostic tool. Here, we compare the most commonly used techniques for the assessment of NET formation. Furthermore, we review recent data from the literature on the application of basic laboratory tools for detecting NET release and discuss the challenges and the advantages of these strategies in NET evaluation. Taken together, we provide some important insights into the qualitative and quantitative molecular analysis of NETs in translational medicine today.

Keywords: neutrophil; neutrophil extracellular traps; techniques; tools.

Figure 1

Schematic diagram demonstrating the different types of NETs. Depending on the stimulation, various molecular pathways result in the generation of NETs. This diagram presents the fundamental pathways of NET production, and in particular, the vital NETs or NADPH oxidase-2 (NOX) -independent pathway and the suicidal NETs or NADPH oxidase-2 (NOX) -dependent pathway. Furthermore, NETs can also consist of mitochondrial DNA (mtDNA) rather than chromosomal DNA. In this pathway, intracellular grey structures represent mitochondrial organelles. Some indicative NET inducers are chemical compounds or inflammatory agents (Phorbol-12-myristate-13-acetate (PMA), antibodies (Abs), lipopolysaccharides (LPS), activated platelets, granulocyte-macrophage colony-stimulating factor (GM-CSF) and complement component C5a).

Figure 2

Immunofluorescence staining: A qualitative tool for NET detection in human-isolated neutrophils using either a confocal or a conventional fluorescence microscope. Neutrophils isolated from healthy donors’ whole blood using a density gradient separation method were stimulated with a serum derived from a treatment-naïve patient with active ulcerative colitis (180 min incubation time). NETs are visualized as fibers of extracellular DNA and neutrophil granule proteins assessed by double-staining with a chromatin-staining dye and a neutrophil marker. (A) Neutrophils were stained with a rabbit anti-human neutrophil elastase ((NE), Abcam, Cambridge, UK) antibody detected by a goat anti-rabbit AlexaFluor 594 antibody (Invitrogen, Waltham, MA, USA). Visualization was performed using confocal microscopy (Revolution spinning disk confocal system; Andor, Belfast, UK). (B) Neutrophils were stained with a mouse anti-human neutrophil elastase ((NE), Abcam) antibody detected by a goat anti-mouse AlexaFluor 488 antibody (Invitrogen). Visualization was performed using a fluorescence microscope (OLYMPUS BX51, Shinjuku, Japan). In (A,B), cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole ((DAPI), Sigma-Aldrich, Burlington, MA, USA). Untreated neutrophils served as control. Data from the Molecular Hematology Laboratory archive are shown.

Figure 3

Immunofluorescence staining: a qualitative tool to detect NET remnants or disease-related proteins located on NETs in human tissue specimens. Cross-sections (4 μm thickness) of kidney biopsy tissue derived from a patient with proliferative lupus nephritis (LN) were deparaffinized and stained with the appropriate antibodies. (A) NETs were visualized in kidney specimens as extracellular structures by staining with both a mouse anti-human neutrophil elastase ((NE), Abcam) antibody and a rabbit anti-human histone 3 (Anti-Histone H3 (citrulline R2 + R8 + R17), Abcam) antibody. (B) The presence of a disease-related protein on extracellular structures, such as IL-17 in LN, was examined using both a mouse anti-human IL-17A (R&D Systems) antibody and a rabbit anti-human histone H3 (Anti-Histone H3 (citrulline R2 + R8 + R17), Abcam) antibody. In (A,B), a goat anti-mouse AlexaFluor 488 antibody (Invitrogen) and a goat anti-rabbit AlexaFluor 594 antibody (Invitrogen) were used to detect primary antibodies. Sections were counterstained with DAPI and visualized in a confocal microscope (Revolution spinning disk confocal system; Andor, Belfast, UK). Data from the Molecular Hematology Laboratory archive are shown.

Figure 4

Flow cytometry assay: an indicative quantitative method of NET determination in human isolated neutrophils. Flow cytometric analysis of human neutrophils derived from a healthy donor. Neutrophils were isolated from whole blood using a density gradient separation method. (A) Unstimulated neutrophils (negative control) or (B) neutrophils stimulated with a chemical inducer of NETs (phorbol 12-myristate 13-acetate (PMA), Sigma-Aldrich; 20 nM final concentration, 45 min incubation time) were stained with a neutrophil activation marker (mouse anti-human CD66b—conjugated with PerCP/Cyanine5.5, Biolegend, San Diego, CA, USA). CD66b positive cells were examined for the presence of citrullinated histone 3 upon staining with a rabbit anti-human histone 3 antibody (Recombinant anti-histone H3 (citrulline R8), Abcam) detected with a goat anti-rabbit AlexaFluor488 antibody (Invitrogen). It was tested that PMA did not induce apoptosis/necrosis in neutrophils under these experimental conditions. Data from the Molecular Hematology Laboratory archive are shown.