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.

Background Histone deimination regulates gene function and contributes to antimicrobial response, allowing the formation of neutrophil extracellular traps (NETs). Deiminated proteins are target of anti-citrullinated peptides antibodies (ACPA) in rheumatoid arthritis (RA). Objective The objective of this paper is to test the hypothesis that RA sera react with deiminated histones contained in NETs. Methods Neutrophils from peripheral blood were stimulated with A23187 and acid treated; NETosis was induced by phorbol myristate acetate, and NET proteins were isolated. Sera were tested by immunoblot on acid extracted proteins from neutrophils and from NETs, and by ELISA on deiminated histone H4 or H4-derived peptides. Bands reactive with RA sera were excised from gels, digested with trypsin and subjected to matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) analysis, before and after derivatisation to detect citrullinated peptides. Results RA sera reacted with a deiminated antigen of 11 KDa from activated neutrophils, recognised also by anti-H4 and antideiminated H4 antibodies. A similar reactivity was observed with NET proteins. The antigen from neutrophils or NETs was identified as citrullinated H4 by MALDI-TOF analysis. By ELISA, RA sera bound in vitro citrullinated H4. Citrullinated H4 14–34 and 31–50 peptides detected antibodies in 67% and 63% of RA sera and in less than 5% of controls; antibody titre was correlated with anti-CCP2. Conclusions Citrullinated H4 from activated neutrophils and NETs is a target of antibodies in RA, and synthetic citrullinated H4-derived peptides are a new substrate for ACPA detection. As NETosis can generate antigens for ACPA, these data suggest a novel connection between innate and adaptive immunity in RA.

Objectives Rheumatoid arthritis (RA) is characterised by breach of self-tolerance towards citrullinated antigens with generation of anti-citrullinated peptide/proteins antibodies (ACPA). Currently, the nature and source of citrullinated antigens driving the humoral autoimmune response within synovial ectopic lymphoid structures (ELS) is a crucial unknown aspect of RA pathogenesis. Here we characterised the autoreactive B-cell response of lesional B cells isolated from ELS+RA synovium. Methods Single synovial tissue CD19+cells were Fluorescence Activated Cell Sorting (FACS)-sorted and V_H/V_L Ig genes cloned to generate recombinant monoclonal antibodies (rmAbs) from patients with ELS+/ACPA+RA. Results RA-rmAbs immunoreactivity analysis provided the following key findings: (1) in a chIP-based array containing 300 autoantigens and in a ‘citrullinome’ multiplex assay, a strong reactivity against citrullinated histones H2A/H2B (citH2A/H2B) was observed in ∼40% of RA-rmAbs, followed by cit-fibrinogen and cit-vimentin; (2) anti-citH2A/H2B-reactive RA-rmAbs (but not anti-citH2A/H2B negative) selectively recognised neutrophil extracellular traps (NETs) from peripheral blood and/or RA joint neutrophils; (3) anti-citH2A/citH2B and anti-NET immunobinding was dependent on affinity maturation and was completely abrogated following reversion of hypermutated IgV_H/V_L genes to germline sequences; (4) ELS+ (not ELS−) RA synovial tissues engrafted into Severe Combined ImmunoDeficiency (SCID) mice released human anti-citH2A/citH2B and anti-NET antibodies in association with the intra-graft expression of CXCL13 and lymphotoxin (LT)-β, two master regulators of ELS. Conclusion We provided novel evidence that B cells differentiated within synovial ELS in the RA joints frequent target deiminated proteins which could be generated during NETosis of RA synovial neutrophils including histones. Thus, NETs could represent a source of citrullinated antigens fuelling the ACPA autoimmune response within the RA synovium.

In neutrophils (but also in eosinophils and in mast cells) different inflammatory stimuli, induce histone deimination, chromatin decondensation and NET formation. These web-like structures that trap and kill microbes contain DNA, cationic granule proteins and anti-microbial peptides, but the most abundant proteins are core histones. Histones contained in NETs have been deiminated and arginines are converted in citrullines. While deimination is a physiological process amplified in inflammatory conditions, only individuals carrying genetic predisposition to develop rheumatoid arthritis (RA) make antibodies to deiminated proteins. These antibodies, collectively identified as anti citrullinated proteins antibodies (ACPA), react with different deiminated proteins and display partially overlapping specificities. In this paper, we will summarize current evidence supporting the role of NETosis as critical mechanism in the breach of tolerance to self-antigens and in supporting expansion and differentiation of autoreactive cells. In fact, several lines of evidence connect NETosis with RA: RA unstimulated synovial fluid neutrophils display enhanced NETosis; sera from RA patients with Felty’s syndrome bind deiminated H3 and NETs; a high number of RA sera bind deiminated H4 contained in NETs; human monoclonal antibodies generated from RA synovial B cells decorate NETs and bind deiminated histones. In RA, NETs represent on one side an important source of autoantigens bearing post-translational modifications and fuelling the production of ACPA. On the other side, NETs deliver signals that maintain an inflammatory milieu and contribute to the expansion and differentiation of ACPA-producing B cells.