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Neutrophil extracellular traps (NET), extruded decondensated chromatin entangled with neutrophil proteases, have been first identified in neutrophils stimulated with bacteria or phorbol myristate acetate (PMA) via activation of NADPH oxidase and the generation of reactive oxygen species. Although the first findings demonstrated the beneficial role of NET formation by trapping the bacteria and limiting their dissemination, numerous studies in the recent decade revealed the multifunctional aspects of NET formation which manifests itself not only in the context of anti-microbial effect but also as a pathological trigger. Uncontrolled and exaggerated NET formation or inability to digest and remove NET have been reported in thrombosis, auto-immune diseases, cancer or even in infertility. Studies are ongoing to disclose the role of NET in different pathological situations and most importantly, NET regulation via compounds that either interfere with NET formation or target NET components such as DNA or neutrophil proteases. Although the final product of NET formation seems to be quite common i.e. DNA entangled with proteases, stimuli that induce NET have a wide range of varieties and the involved pathways are diverse too. Therefore, in every pathological condition, it is necessary to consider carefully the type of stimulus and the signaling pathways in order to target the disease more specifically. Here we briefly summarize some (out of many) NET triggers/pathways and discuss the potential interventions in the pathological situations.

Neutrophils, as the first cellular line of innate host defense, employ phagocytosis and formation of neutrophil extracellular traps (NETs) to combat infections. Classical NET formation induced by phorbol myristate acetate requires several hours to complete. However, recent studies demonstrated rapid NET formation in neutrophils upon stimulation by platelets, Staphylococcus aureus or fungal products. Here we describe that antibody- or complement-induced phagocytosis triggers rapid NET formation. In contrast to classical NETosis, chemical inhibition of NADPH oxidase as well as using NADPH oxidase-deficient patient neutrophils did not affect rapid NET formation. Although phagocytosis and rapid NET formation may not be the prerequisite of each other, cooperation of phagocytosis and rapid NET formation may be essential to improve the efficiency of defense mechanisms in combating disseminating bacteria. Dissecting the differential mechanisms of NET formation is crucial to develop novel therapeutic strategies for infectious and auto-immune diseases where NETs play an essential role.

Expanding evidence indicates multiple interactions between the hemostatic system and innate immunity, and the coagulation and complement cascades. Here we show in a tissue factor (TF)-dependent model of flow restriction-induced venous thrombosis that complement factors make distinct contributions to platelet activation and fibrin deposition. Complement factor 3 (C3) deficiency causes prolonged bleeding, reduced thrombus incidence, thrombus size, fibrin and platelet deposition in the ligated inferior vena cava, and diminished platelet activation in vitro. Initial fibrin deposition at the vessel wall over 6 hours in this model was dependent on protein disulfide isomerase (PDI) and TF expression by myeloid cells, but did not require neutrophil extracellular trap formation involving peptidyl arginine deiminase 4. In contrast to C3-/- mice, C5-deficient mice had no apparent defect in platelet activation in vitro, and vessel wall platelet deposition and initial hemostasis in vivo. However, fibrin formation, the exposure of negatively charged phosphatidylserine (PS) on adherent leukocytes, and clot burden after 48 hours were significantly reduced in C5-/- mice compared with wild-type controls. These results delineate that C3 plays specific roles in platelet activation independent of formation of the terminal complement complex and provide in vivo evidence for contributions of complement-dependent membrane perturbations to prothrombotic TF activation on myeloid cells.

Mesenchymal stem cells (MSCs) are crucial for tissue homeostasis and regeneration. Though of prime interest, their potentially protective role on neutrophil-induced tissue damage, associated with high morbidity and mortality, has not been explored in sufficient detail. Here we report the therapeutic skill of MSCs to suppress unrestrained neutrophil activation and to attenuate severe tissue damage in a murine immune-complex mediated vasculitis model of unbalanced neutrophil activation. MSC-mediated neutrophil suppression was due to intercellular adhesion molecule 1-dependent engulfment of neutrophils by MSCs, decreasing overall neutrophil numbers. Similar to MSCs in their endogenous niche of murine and human vasculitis, therapeutically injected MSCs via upregulation of the extracellular superoxide dismutase (SOD3), reduced superoxide anion concentrations and consequently prevented neutrophil death, neutrophil extracellular trap formation and spillage of matrix degrading neutrophil elastase, gelatinase and myeloperoxidase. SOD3-silenced MSCs did not exert tissue protective effects. Thus, MSCs hold substantial therapeutic promise to counteract tissue damage in conditions with unrestrained neutrophil activation.

The extracellular adherence protein (Eap) of Staphylococcus aureus is a secreted protein known to exert a number of adhesive and immunomodulatory properties. Here we describe the intrinsic DNA binding activity of this multifunctional secretory factor. By using atomic force microscopy, we provide evidence that Eap can bind and aggregate DNA. While the origin of the DNA substrate (e.g., eukaryotic, bacterial, phage, and artificial DNA) seems to not be of major importance, the DNA structure (e.g., linear or circular) plays a critical role with respect to the ability of Eap to bind and condense DNA. Further functional assays corroborated the nature of Eap as a DNA binding protein, since Eap suppressed the formation of "neutrophil extracellular traps" (NETs), composed of DNA-histone scaffolds, which are thought to function as a neutrophil-mediated extracellular trapping mechanism. The DNA binding and aggregation activity of Eap may thereby protect S. aureus against a specific anti-microbial defense reaction from the host.

In the recent decade, neutrophil extracellular traps (NETs) have been identified and confirmed as a new anti-microbial weapon of neutrophils. In this protocol, we describe easy methods to demonstrate NET formation by immunofluorescence staining of extracellular chromatin fiber with anti-DNA/Histone H1 antibody and quantification of NETs by using a non-cell-permeable DNA specific dye Sytox orange.

Deficiencies in many coagulation factors and protease-activated receptors (PARs) affect mouse embryonic development, but phenotypes of single PAR-deficiencies are often obscured by compensation by other receptors. Although most PAR1/PAR2 double deficient embryos do not survive to birth, PAR2 single-deficient embryos exhibit an unresolved partial preterm lethality. We have observed that PAR2-/- embryos at day 13.5 to 15.5 p.c. from timed mating of homozygous PAR2-/- parents display an increased embryonic death and growth retardation as well as variable signs of anemia in comparison to WT embryos of the same gestational age. The pale appearance suggested that PAR2 might play a role during fetal hematopoiesis. To investigate whether this phenotype is merely a consequence of a previously demonstrated placental defect arising from PAR2 deficiency in the mother, we compared heterozygous PAR2+/- embryos produced by timed-mating of WT mothers mated with PAR2-/- fathers or of PAR2-/- mother mated with WT father. Although reduced embryo weights in PAR2-/- relative to WT mothers indicated a developmental delay due to maternal PAR2 deficiency, anemia was not observed in these PAR2+/- embryos. The fetal liver is the site of hematopoiesis during embryonic development. We therefore evaluated fetal hematopoiesis at day 13.5 to 15.5 p.c. in PAR2+/- and PAR2-/- embryos from timed mated heterozygous PAR2+/- mothers mated with PAR2-/- fathers. Macroscopic phenotypic abnormalities, including signs of anemia, were more prevalent in PAR2-/- versus PAR2+/- embryos, demonstrating an embryonic phenotype that develops independent from maternal PAR2-deficiency. Gene expression analyses of fetal liver at day 13.5 p.c. showed a reduction in marker genes for erythropoiesis, i.e. beta-globin, GATA-1, BCL-11a and for macrophages, i.e. F4/80, Id3, Nr1h3, specifically in PAR2-/- relative to PAR2+/- embryos. These differences were also seen at day 15.5 p.c. in homozygous PAR2 R38E cleavage resistant relative to PAR2 R38E/WT heterozygous embryos in the same litter, confirming PAR2 proteolytic signaling as the major regulator of fetal hematopoiesis. Since macrophage markers in the fetal liver were reduced in PAR2-/- embryos, we next evaluated the role of macrophage PAR2 signaling by cell-type specific deletion in PAR2flfl-LysMcre mice. Macrophage PAR2-deficient mice had reduced erythroblast islands, and erythropoiesis and macrophage marker genes were reduced in the fetal liver at day 15.5 p.c. to the same extent as seen in completely PAR2-deficient embryos. Blood island macrophages play pivotal roles in erythrophagocytosis and clearance of red blood cell expelled nuclei. Deficiency in red blood cell nuclei clearance, e.g. in DNase I deficient mice, causes macrophage inflammatory activation and upregulation of the interferon regulated gene Hamp-1. PAR2flfl-LysMcre fetal livers showed increased Hamp-1 expression relative to cre-negative controls from the same pregnancy, further indicating that macrophage PAR2 signaling directly participates in the regulation of erythropoiesis. These data identify a novel function for proteolytic PAR2 activation in the supporting fetal hematopoiesis and raise the possibility that PAR2 signaling may also regulate adult hematopoiesis under stress conditions or aging. Disclosures No relevant conflicts of interest to declare.

Neutrophils, the most abundant human immune cells, are rapidly recruited to sites of infection, where they fulfill their life-saving antimicrobial functions. While traditionally regarded as short-lived phagocytes, recent findings on long-term survival, neutrophil extracellular trap (NET) formation, heterogeneity and plasticity, suppressive functions, and tissue injury have expanded our understanding of their diverse role in infection and inflammation. This review summarises our current understanding of neutrophils in host-pathogen interactions and disease involvement, illustrating the versatility and plasticity of the neutrophil, moving between host defence, immune modulation, and tissue damage.

Posttranslational modification of the neural cell adhesion molecule (NCAM) by polysialic acid (polySia) is well studied in the nervous system and described as a dynamic modulator of plastic processes like precursor cell migration, axon fasciculation, and synaptic plasticity. Here, we describe a novel function of polysialylated NCAM (polySia-NCAM) in innate immunity of the lung. In mature lung tissue of healthy donors, polySia was exclusively attached to the transmembrane isoform NCAM-140 and located to intracellular compartments of epithelial cells. In patients with chronic obstructive pulmonary disease, however, increased polySia levels and processing of the NCAM carrier were observed. Processing of polysialylated NCAM was reproduced in a mouse model by bleomycin administration leading to an activation of the inflammasome and secretion of interleukin (IL)-1β. As shown in a cell culture model, polySia-NCAM-140 was kept in the late trans-Golgi apparatus of lung epithelial cells and stimulation by IL-1β or lipopolysaccharide induced metalloprotease-mediated ectodomain shedding, resulting in the secretion of soluble polySia-NCAM. Interestingly, polySia chains of secreted NCAM neutralized the cytotoxic activity of extracellular histones as well as DNA/histone-network-containing "neutrophil extracellular traps", which are formed during invasion of microorganisms. Thus, shedding of polySia-NCAM by lung epithelial cells may provide a host-protective mechanism to reduce tissue damage during inflammatory processes.

SummaryThe functions of extracellular RNA in the vascular system as new procoagulatory and permeability-increasing factor in vivoand in vitrowere shown to be counteracted by pancreatic type RNase1. Based on the identification of RNase1 in plasma and serum, it is proposed that the enzyme is expressed by vascular cells to contribute in the regulation of extracellular RNA. It is demonstrated that RNase1 and RNase5 (also termed angiogenin) were differentially expressed in various types of endothelial cells, whereby human umbilical vein endothelial cells (HUVEC) expressed and released the highest concentration of active RNase1. Expression and release of RNase5 were similar in all types of endothelial cells tested. Both RNases were constitutively produced and secreted, whereby a portion of RNase1, but not RNase5, was stored in Weibel-Palade bodies, co-localising with von Willlebrand factor and P-selectin. Accordingly, immediate release of RNase1 from these granules was demonstrated in vitroand in vivousing Weibel-Palade body exocytosis-inducing agents. Additionally, extracellular RNA or poly:IC (but not DNA) induced this short-term release of RNase1. Our results indicate that vascular RNase1 and RNase5 are mainly produced by vascular endothelial cells and can serve, depending on the vascular bed, different functions in vascular homeostasis and endothelial cell responses.