Neutrophil extracellular traps in inflammatory disorders

A novel feature of polymorphonuclear granulocyte (PMN) biology is their ability to generate neutrophil extracellular traps (NETs) via a distinct process of cell death termed NETosis. NETs consist of chromosomal extruded DNA decorated with granular components that include antimicrobial peptides and proteases. The molecular pathways leading to NETosis involve generation of reactive oxygen species (ROS), calcium mobilization, nuclear delobulation involving the enzymatic activities of myeloperoxidase (MPO) and neutrophil elastase (NE), and chromatin modification via the citrullination of histones by peptidyl arginine deiminase 4 (PAD4). A number of studies have implicated NETs in the etiology of auto-inflammatory or autoimmune conditions such as preeclampsia (PE), Felty’s syndrome, systemic lupus erythematosus (SLE), multiple sclerosis (MS), and, most recently, rheumatoid arthritis (RA). In the context of RA these findings are especially interesting, as NETs have been proposed to contribute to the generation of anti-citrullinated protein antibody (ACPA) auto-antigens, and may also be a target for autoantibodies. As we have previously detected significantly increased concentrations of cell-free DNA in the sera of RA patients compared with healthy controls, we were intrigued whether the elevation in this material involved NETosis. The premise for this investigation was that a link between circulating cell-free DNA levels and NETs has previously been made in a number of conditions including preeclampsia, sepsis, cancer, thrombosis or even storage of blood transfusion products. In view of these findings and reports on the complex involvement of neutrophil NETs in autoimmunity, we sought to investigate the NETotic response of PMN in RA, with particular regard to the underlying signal transduction cascade, and whether the products of overt NETosis could be diagnostically useful. Our data confirm and extend upon these observations in that we examined for changes in the underlying signal transduction cascade required for the induction of NETosis. These findings indicate that the propensity of circulatory PMN in RA patients to undergo NETosis is associated with elevations in members of this cascade including increased intracellular ROS production, enhanced expression of NE and MPO, and increased nuclear translocation of PAD4 and citrullination of histones, notably H3. Furthermore, by examining kinetic changes during extended in vitro culture it was observed that PMN from RA cases exhibited different nuclear morphometric characteristics, having a lower proportion of the classical lobulated phenotype. In the presence of a secondary stimuli, RA PMN also progressed more rapidly and extensively to a NETotic spread phenotype than controls, a finding confirmed by analysis of culture supernatants for the products of NETosis. Akin to what has been observed in an array of other pathological conditions ranging from SLE to cancer, PMN from RA patients exhibited an increased response to further stimulation. This response is in part mediated via the action of PAD4, as the effect of PMA could be significantly reduced by treatment with Cl-amidine, an inhibitor of PAD4. In addition, PMA treatment lead to an increased nuclear presence of this enzyme, where it presumably could be able to carry out a more extensive citrullination of histone proteins, thereby speeding up the induction of NETosis. A very intriguing and novel feature of our data is the detection of the extracellular presence of PAD4 on extruded NETs. The concomitant extracellular presence of this enzyme on NETs may increase the spectrum of citrullinated proteins and their local concentration significantly, a feature which could be important in the generation of ACPA. It is furthermore possible that the extracellular localisation of PAD4 may play a role in the development of anti-PAD4 antibodies observed in cases with RA. Hence, our data support and extend upon recent reports indicating that NETs can be a source for citrullinated autoantigens, or react with ACPA. Further presence of NETs in the intervillous space of preeclamptic placenta was previously reported by our lab. Using in vitro experiments, we have shown that trophoblast micro-debris could stimulate neutrophils to form NETs. However the effect of such phenomena remained elusive. Recent data suggests, NETs could act as a lattice, to stimulate platelet adhesion, promotes coagulation and thrombotic events. Given that PE, intrauterine growth restriction (IUGR) and even fetal loss are broadly related to elevated thrombotic events causing dysfunctions at the interface between innate immunity and haemostasis, it is of cardinal importance to investigate whether NETs could elucidate such a response. In this study, we adopted mice sFLT-1 overexpression model, to elucidate the impact of NETosis in murine pregnancy outcome. Preliminary results indicates significant drop in pregnancies after sFLT-1 overexpression although there was only a partial induction of PE phenotype characterized by minor elevation in blood pressure and proteinuria. Staining of placentas from mice that retained pregnancy after sFLT-1 overexpression indicates heavy neutrophil infiltration in the feto-placental junction, with zones positive for NET specific markers. Plasma isolated from sFLT-1 treated mouse also indicates elevation of NET specific markers compared to healthy pregnant controls. Minor increase in plasma TAT complexes (although not significant), along with a drop in platelet count, indicates elevated thrombotic events. Further ongoing experiments will compare the impact of sFLT-1 overexpression in normal WT mice to those that are unable to form NETs.