Introduction: Neutrophil extracellular traps (NETs) have recently been implicated in a number of autoimmune conditions, including rheumatoid arthritis (RA). We examined the underlying signaling pathways triggering enhanced NETosis in RA and ascertained whether the products of NETosis had diagnostic implications or usefulness. Methods: Neutrophils were isolated from RA patients with active disease and from controls. Spontaneous NET formation from RA and control neutrophils was assessed in vitro with microscopy and enzyme-linked immunosorbent assay (ELISA) for NETosis-derived products. The analysis of the signal-transduction cascade included reactive oxygen species (ROS) production, myeloperoxidase (MPO), neutrophil elastase (NE), peptidyl arginine deiminase 4 (PAD4), and citrullinated histone 3 (citH3). NET formation was studied in response to serum and synovial fluid and immunoglobulin G (IgG) depleted and reconstituted serum. Serum was analyzed for NETosis-derived products, for which receiver operator characteristic (ROC) curves were calculated. Results: Neutrophils from RA cases exhibited increased spontaneous NET formation in vitro, associated with elevated ROS production, enhanced NE and MPO expression, nuclear translocation of PAD4, PAD4-mediated citrullination of H3, and altered nuclear morphology. NET formation in both anti-citrullinated peptide antibody (ACPA)-positive and -negative RA was abolished by IgG depletion, but restored only with ACPA-positive IgG. NETosis-derived products in RA serum demonstrated diagnostic potential, the ROC area under the curve for cell-free nucleosomes being >97%, with a sensitivity of 91% and a specificity of 92%. No significant difference was observed between ACPA-positive and -negative cases. Conclusions: Signaling elements associated with the extrusion of NETs are significantly enhanced to promote NETosis in RA compared with healthy controls. NETosis depended on the presence of ACPA in ACPA-positive RA serum. The quantitation of NETosis-derived products, such as cell-free nucleosomes in serum, may be a useful complementary tool to discriminate between healthy controls and RA cases.

Human pregnancy is associated with a mild pro-inflammatory state, characterized by circulatory neutrophil activation. In order to explore the mechanism underlying this alteration, we examined NETosis during normal gestation. Our data indicate that neutrophils exhibit a pro-NETotic state, modulated in a multimodal manner during pregnancy. In general, circulatory granulocyte colony-stimulating factor, the levels of which increase during gestation, promotes neutrophil extracellular trap (NET) formation. Early in pregnancy, NETosis is enhanced by chorionic gonadotropin, whereas toward term is stimulated by estrogen. A complex interaction between estrogen and progesterone arises, wherein progesterone restrains the NETotic process. In this state, extensive histone citrullination is evident, yet full NETosis is inhibited. This coincides with the inability of neutrophil elastase to translocate from the cytoplasm to the nucleus and is regulated by progesterone. Our findings provide new insight concerning gestational and hormone-driven pathologies, since neutrophil recruitment, activation, and NET release could be associated with excessive endothelial and placental injury.

The ability of neutrophils and other leucocyte members of the innate immune system to expel their DNA into the extracellular environment in a controlled manner in order to trap and kill pathogenic microorganisms lead to a paradigm shift in our understanding of host microbe interactions. Surprisingly, the neutrophil extracellular trap (NET) cast by neutrophils is very wide and extends to the entrapment of viruses as well as multicellular eukaryotic parasites. Not unexpectedly, it has emerged that pathogenic microorganisms can employ a wide array of strategies to avoid ensnarement, including expression of DNAse enzymes that destroy the lattice backbone of NETs. Alternatively, they may use molecular mimicry to avoid detection or trigger events leading to the expression of immune modulatory cytokines such as IL-10, which dampen the NETotic response of neutrophils. In addition, the host microenvironment may contribute to the innate immune response by the production of lectin-like molecules that bind to bacteria and promote their entrapment on NETs. An example of this is the production of surfactant protein D by the lung epithelium. In addition, pregnancy provides a different challenge, as the mother needs to mount an effective response against pathogens, without harming her unborn child. An examination of these decoy and host response mechanisms may open the path for new therapies to treat pathologies mediated by overt NETosis.

Introduction: The analysis of cell-free DNA from maternal blood samples has facilitated the noninvasive detection of fetal aneuploidies or hereditary Mendelian disorders. In this context, previous studies have indicated that the pool of cell-free DNA is greater in maternal serum than in plasma samples, necessitating optimized collection and storage protocols. As the source of this increased amount of cell-free DNA is not clear, we have now examined whether neutrophil extracellular traps (NETs) contribute to this material. Material and Methods: Serum samples were collected in all three trimesters of normal healthy pregnant women, and at term from cases with manifest preeclampsia. The presence of NET-derived material was demonstrated by the detection of cell-free DNA fragments complexed to neutrophil granular proteins (i.e. myeloperoxidase). Results: Our data indicate that NET-derived cell-free DNA/myeloperoxidase complexes were greater in serum from normal pregnant women than in normal matching nonpregnant controls. This neutrophil chromosomal material increased incrementally throughout gestation and was most pronounced in cases with preeclampsia. Discussion: By detecting increased levels of cell-free DNA/myeloperoxidase complexes in maternal serum samples, our data indicate that a significant proportion of this material is derived from the generation of NETs.

Inflammation is thought to play a critical role in the pathogenesis of placentation disorders such as recurrent miscarriages, growth restriction, and

Pregnancy is associated with elevated maternal levels of cell-free DNA of neutrophil extracellular trap (NET) origin, as circulatory neutrophils exhibit increased spontaneous NET formation, mainly driven by G-CSF and finely modulated by sex hormones. The postpartum period, on the other hand, involves physiological alterations consistent with the need for protection against infections and fatal haemorrhage. Our findings indicate that all relevant serum markers of neutrophil degranulation and NET release are substantially augmented postpartum. Neutrophil pro-NETotic activity in vitro is also upregulated particularly in post-delivery neutrophils. Moreover, maternal puerperal neutrophils exhibit a strong pro-NETotic phenotype, associated with increased levels of all key players in the generation of NETs, namely citH3, MPO, NE, and ROS, compared to non-pregnant and pregnant controls. Intriguingly, post-delivery NET formation is independent of G-CSF in contrast to late gestation and complemented by the presence of TF on the NETs, alterations in the platelet activity status, and activation of the coagulation cascade, triggered by circulating microparticles. Taken together, our results reveal the highly pro-NETotic and potentially procoagulant nature of postpartum neutrophils, bridging an overt immune activation with possible harmful thrombotic incidence.