Abstract
Neutrophil extracellular traps (NET) formation is part of the neutrophil response to infections, but excessive or inappropriate NETosis may trigger the production of autoantibodies and cause organ damage in autoimmune disorders. Spontaneously netting neutrophils are not frequent and induction of NET in vitro by selected stimuli is necessary to investigate their structure. In the present work, the protein composition and post-translational modifications of NET produced under different stimuli have been studied by means of proteomic analysis. Neutrophils from healthy donors were stimulated by PMA, A23187, Escherichia coli LPS or untreated; after three hours, cells were washed, treated with DNase and supernatants collected for mass spectrometry. Data were analyzed by unsupervised hierarchical clustering analyses. We identified proteins contained in NETs of any source or exclusive of one stimulus: LPS-induced and spontaneous NET diverge in protein composition, while PMA- and A23187-induced NET appear more similar. Among the post-translational modifications we examined, methionine sulfoxidation is frequent especially in PMA- and LPS-induced NETs. Myeloperoxidase is the protein more extensively modified. Thus, proteomic analysis indicates that NETs induced by different stimuli are heterogeneous in terms of both protein composition and post-translational modifications, suggesting that NET induced in different conditions may have different biological effects.
Highlights
Neutrophil extracellular traps (NET) formation (NETosis) is a mechanism of defense that neutrophils deploy as an alternative to phagocytosis, to constrain the spread of fungi, large bacteria and several other microorganisms [1,2]
74 proteins (22%) were detected in NETs of any source while 30 (9.1%), 7 (2.1%), 27 (8.2%) and 40 (12.1%) proteins were exclusive, respectively, of spontaneous NETs, or NETs obtained with LPS, A23187 and phorbol myristate acetate (PMA) (Fig 1A and S1 Table)
No major differences are observed in the frequency of the other Post translational modifications (PTM) we studied, but individual proteins bearing PTM may be differentially enriched in NET obtained with the various stimuli: oxidized MPO and cathepsin G are abundant in PMA- and A23187-induced NET, oxidized calprotectin in A23187- and LPS-induced NET, while oxidized actin is highly represented in A23187-induced NET
Summary
Neutrophil extracellular traps (NET) formation (NETosis) is a mechanism of defense that neutrophils deploy as an alternative to phagocytosis, to constrain the spread of fungi, large bacteria and several other microorganisms [1,2]. During NETosis, nucleus decondenses and intracellular membranes disintegrate; nuclear and cytoplasmic content mixes and plasma membrane. Comparative proteomic analysis of NETs permeabilizes, allowing the extrusion of a tangle of chromatin fibers decorated with granule proteins. In the best characterized pathway leading to NET extrusion, ERK signaling leads to the activation of NADPH oxidase and the production of superoxide radicals that are converted into oxygen peroxide by superoxide dismutase [3]. NE is responsible for degradation of cytoskeleton and nuclear membrane, allowing the mixing of nuclear content and cytoplasm [4]. Histone deimination by activated peptidylarginine deiminase (PAD) and proteolytic cleavage by MPO and NE lead to chromatin decondensation [5]. Chromatin fibers associate with granule and cytoplasmic proteins and are released extracellularly. Transcriptional firing at multiple locations contributes to chromatin decondensation [6], but NET formation proceeds independently of translation [7]
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