NETosis as Source of Autoantigens in Rheumatoid Arthritis

Abstract

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 antimicrobial 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/peptides 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 posttranslational modifications and fueling 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.

Keywords: autoantibodies; autoantigens; ectopic lymphoid structures; histones; neutrophils extracellular traps; rheumatoid arthritis.

Figure 1

A high number of citrullinated proteins is produced in neutrophils upon stimulation. Total proteins from A23187-stimulated (S) or unstimulated (NS) neutrophils were subjected to SDS-PAGE and western blotting and probed with an anti-modified citrulline human monoclonal antibody (anti-MC), NHS or RA sera, anti-citrullinated H3 (anti-H3cit), or anti-citrullinated H4 (anti-H4cit) polyclonal antibodies. Anti-MC h-mAb decorates a high number of proteins in stimulated neutrophils, part of which are recognized by RA sera. Proteins of 10–15 KDa are bound by anti-MC h-mAb, by RA sera, and by anti-H4cit or anti-H3cit antisera, thus suggesting that in stimulated neutrophils, H3 and H4 are citrullinated and become target of RA immune response, as previously showed (, with the permission of BMJ Publishing Group, N° 3976570562524).

Figure 2

Neutrophil NETosis and anti-NET antibodies in synovial ELS: a new model suggesting linking inflammation and autoimmunity to citrullinated proteins in RA. The figure illustrates the hypothetical evolution of ACPA from a pre-disease phase outside the joint to a disease phase associated with chronic synovial inflammation. In particular, it has been proposed that environmental factors such as bacterial infection in periodontal disease and smoking can lead to the formation of citrullinated antigens outside the joint which, in the context of HLA-DRB1 shared epitope, can lead to the production of ACPA prior to the clinical onset. However, the second inflammatory hit that occurs in the synovium is not clear yet. Here, we proposed that migration of neutrophils from the periphery followed by aberrant neutrophil NETosis during chronic inflammation within the RA compartment (synovial tissue and fluid) provide a continuous source of externalized citrullinated antigens, such as citH2A, citH2B, and citH4 histones that can be presented by antigen-presenting cells following engulfment of neutrophils nuclear fragments. Such process would sustain an antigen-driven autoimmune response toward citrullinated antigens within ELS in the RA joint resulting in the production of anti-NET autoantibodies, which may contribute to the perpetuation of chronic inflammation and autoimmunity (75).