Session: Session 1

Proteomic Dissection of Neutrophil Extracellular Traps (NETs): Unveiling the Molecular Landscape

Magalie DUCHATEAU¹, Quentin GIAI GIANETTO^1,2, Vanessa KREMER^3,4, Anne DANCKAERT⁵, Pierre BRUHNS³, Friederike JÖNSSON^3,6, Luc DE CHAISEMARTIN^3,4,7, Mariette MATONDO¹

¹Institut Pasteur, Université Paris-Cité, Mass Spectrometry for Biology (MSBio) UtechS, CNRS UAR 2024, Paris, France
² Institut Pasteur, Université Paris Cité, Bioinformatics Hub, Paris, France
³Institut Pasteur, Université Paris-Cité, INSERM UMR1222, Antibodies in Therapy and Pathology, Paris, France
⁴Inflammation, Microbiome and Immunosurveillance, INSERM, Université Paris-Saclay, Orsay, France
⁵Institut Pasteur, Université Paris-Cité, UTechS BioImaging-C2RT, Paris, France
⁶CNRS, Paris, France
⁷APHP, Bichat hospital, Immunology Department, Paris, France

Neutrophils possess a versatile array of antimicrobial mechanisms, with one of the extreme being the formation of Neutrophil Extracellular Traps (NETs). This process involves the release of their own DNA, adorned with cytoplasmic and granule-derived proteins, to trap and neutralize pathogens. NETs have garnered significant interest due to their involvement in various physiological and pathological processes including cancer. Although numerous stimuli have been reported to induce NET formation (NETosis), reproducibility has often been an issue, hindering a comprehensive understanding of NET activation mechanisms. To advance our knowledge, we examined the effects of different stimuli on NET formation and analyzed the proteome.

We selected 6 representative stimuli: phorbol 12-myristate 13-acetate, calcium ionophore A23187, panton-valentine leukocidin toxin, peptidoglycan, TNF-α, and heme. Following stimulation, proteins were resuspended in 1% SDS, reduced and alkylated. Proteins isolation and digestion were performed using the SP3 method. Resulting peptides were cleaned using AssayMAP C18 cartridges and analyzed by DDA on an Orbitrap Q Exactive Plus. The raw data were searched using MaxQuant, and statistical analysis was conducted using our developed R package to identify significant proteins.

More than 3000 proteins were identified with 465 proteins predominantly associated with azurophilic granules and DNA binding functions. Fingerprint proteins were identified for each stimulus, indicating unique NET-associated signatures. Additionally, specific post-translational modifications, such as citrullination and acetylation, were uniquely linked to specific stimuli.

This analysis has elucidated critical components involved in NET formation under various physiological and pathological conditions. These findings provide insights into the molecular mechanisms underlying NETosis and highlight potential targets for therapeutic interventions in NET-associated diseases.