D1.123 - Visualization of neutrophil extracellular traps (NETs) and autoantibodies targeting myeloperoxidase (MPO) and neutrophil elastase (NE) in sputum by confocal microscopy

Neutrophil extracellular traps (NETs) consist of extracellular chromatin (web-like extracellular DNA structures) coated with granule-derived proteins, such as myeloperoxidase (MPO) and neutrophil elastase (NE), and act as a source of autoantigens. Previous studies have reported the presence of autoantibodies directed against NET components and have shown that, particularly in autoimmune and inflammatory diseases, such autoantibodies can promote NET formation, supporting a bidirectional relationship between humoral autoimmunity and NETosis. However, the binding of autoantibodies to NETs has not been directly investigated in asthma patients. We are investigating the role of autoantibodies in driving airway inflammation in severe asthma. Our preliminary data indicate that patients with asthma but not healthy controls have serum autoantibodies that bind to NETs. This abstract presents a method to visualize NETs in sputum samples and quantify their spatial distribution.

Freshly collected sputum was homogenised with 0.1% dithiothreitol, followed by dilution with Dulbecco’s Phosphate Buffered Saline (DPBS) to terminate digestion. Samples were centrifuged, supernatants collected for soluble analyses, and cell pellets resuspended in DPBS for cytospin step. Cells were cytocentrifuged onto glass slides, air-dried, and fixed in 4% paraformaldehyde. Slides were incubated with primary antibodies against MPO and NE, and subsequently with fluorophore-conjugated secondary antibodies. Extracellular DNA was counterstained with 4',6-diamidino-2-phenylindole, and NET structures were visualised (Figure 1A) and quantified using a Nikon confocal microscope and to be analysed with co-localization metrics and statistical tests.

Confocal immunofluorescence analysis revealed a high degree of spatial overlap between MPO and NE along extracellular DNA structures consistent with NETs (Figure 1B). Both antigens localized to identical filamentous and web-like regions, producing superimposable fluorescence patterns.

The co-localisation of MPO and NE on extracellular DNA confirms the presence of NETs in sputum. This immunofluorescence-based approach offers a robust platform for in situ visualization of NET-associated proteins in the airway and mapping their spatial distribution in patients with severe asthma. We are developing a modified protocol based on this method to enable the direct detection of autoantibody binding to NET components.