Neutrophil Extracellular Traps Generation Research Articles

Induction of neutrophil extracellular traps by Campylobacter jejuni.

Campylobacter jejuni is the leading cause of bacterial-derived gastroenteritis worldwide and can lead to several post-infectious inflammatory disorders. Despite the prevalence and health impacts of the bacterium, interactions between the host innate immune system and C. jejuni remain poorly understood. To expand on earlier work demonstrating that neutrophils traffic to the site of infection in an animal model of campylobacteriosis, we identified significant increases in several predominantly neutrophil-derived proteins in the faeces of C. jejuni-infected patients, including lipocalin-2, myeloperoxidase and neutrophil elastase. In addition to demonstrating that these proteins significantly inhibited C. jejuni growth, we determined they are released during formation of C. jejuni-induced neutrophil extracellular traps (NETs). Using quantitative and qualitative methods, we found that purified human neutrophils are activated by C. jejuni and exhibit signatures of NET generation, including presence of protein arginine deiminase-4, histone citrullination, myeloperoxidase, neutrophil elastase release and DNA extrusion. Production of NETs correlated with C. jejuni phagocytosis/endocytosis and invasion of neutrophils suggesting that host- and bacterial-mediated activities are responsible for NET induction. Further, NET-like structures were observed within intestinal tissue of C. jejuni-infected ferrets. Finally, induction of NETs significantly increased human colonocyte cytotoxicity, indicating that NET formation during C. jejuni infection may contribute to observed tissue pathology. These findings provide further understanding of C. jejuni-neutrophil interactions and inflammatory responses during campylobacteriosis.

Enhanced NETosis generation in radiographic axial spondyloarthritis: utility as biomarker for disease activity and anti-TNF-\u03b1 therapy effectiveness

BackgroundRadiographic axial spondyloarthritis (r-axSpA) is a chronic inflammatory form of arthritis in which tumor necrosis factor (TNF)-α, a potent inducer of inflammatory response and a key regulator of innate immunity and of Th1 immune responses, plays a central role. NETosis is a mechanism of innate immune defense that is involved in diverse rheumatology diseases. Nevertheless, spontaneous NETosis generation in r-axSpA, its association to disease pathogenesis, and the NETosis involvement on anti-TNF-α therapy’s effects has never been explored.MethodsThirty r-axSpA patients and 32 healthy donors (HDs) were evaluated. Neutrophil extracellular trap (NET) formation, mediators of signal-transduction cascade required for NETosis induction and cell-free NETosis-derived products were quantified. An additional cohort of 15 r-axSpA patients treated with infliximab (IFX) for six months were further analyzed. In vitro studies were designed to assess the effects of IFX in NETosis generation and the inflammatory profile triggered.ResultsCompared to HDs, neutrophils from r-axSpA patients displayed augmented spontaneous NET formation, elevated expression of NET-associated signaling components, nuclear peptidylarginine deiminase 4 translocation and increased citrullinated histone H3. Furthermore, patients exhibited altered circulating levels of cell-free NETosis-derived products (DNA, nucleosomes and elastase). Additional studies revealed that cell-free NETosis-derived products could be suitable biomarkers for distinguish r-axSpA patients from HDs. Correlation studies showed association between cell-free NETosis-derived products and clinical inflammatory parameters. Besides, nucleosomes displayed potential as a biomarker for discriminate patients according to disease activity.IFX therapy promoted a reduction in both NETosis generation and disease activity in r-axSpA patients. Mechanistic in vitro studies further unveiled the relevance of IFX in reducing NET release and normalizing the augmented inflammatory activities promoted by NETs in mononuclear cells.ConclusionsThis study reveals that NETosis is enhanced in r-axSpA patients and identifies the NETosis-derived products as potential disease activity biomarkers. In addition, the data suggests the potential role of NET generation analysis for assessment of therapeutic effectiveness in r-axSpA.

E‐Cigarette Use Increases Susceptibility to Bacterial Infection by Impairment of Human Neutrophil Chemotaxis, Phagocytosis and NET Formation

Although E‐cigarettes are frequently portrayed as a safer alternative to conventional cigarettes, a growing body of evidence suggests that the use of these devices may adversely affect host defense against infection. Neutrophils, critical effector cells of the innate immune system and early responders to sites of infection, may represent a particularly important cellular target of E‐cigarette vapor components. Using primary human neutrophils isolated from healthy donors, we evaluated the impact of electronic vapor extract (EVE) exposure on multiple aspects of neutrophil cellular physiology, including chemotaxis, reactive oxygen species (ROS) production, generation of Neutrophil Extracellular Traps (NETs) and phagocytosis. Using a transwell chamber approach, we found that exposure to E‐cigarette vapor‐infused media (EVE) reduced neutrophil chemotaxis in response to the chemoattractant f‐Met‐Leu‐Phe (fMLP) by up to 4.2 fold (p < 0.001). Staining with the fluorescent actin probe rhodamine phalloidin revealed aberrant actin cytoskeleton polarization in EVE‐treated, fMLP‐stimulated neutrophils. Neutrophil membrane fluidity was also altered by EVE exposure, with holotomographic microscopy revealing alterations in cellular morphology in EVE‐treated neutrophils. ROS production was reduced in EVE‐treated neutrophils, and ROS‐dependent production of bactericidal extracellular traps was reduced by up to 3.5 fold (p < 0.01). Interestingly, this reduction in NET production (NETosis) was observed in neutrophils treated with EVE produced using propylene glycol‐based E‐cigarette liquid formulations both with and without nicotine. The rate of neutrophil phagocytosis, quantified using fluorescent E. coli/S. aureus bioparticles, was also reduced by up to 47% in EVE‐treated neutrophils (p < 0.05). To determine how such alterations of immune cell function might affect host response to infection, a physiologic mouse model of chronic E‐cigarette exposure and sepsis was utilized. In this model, E‐cigarette vapor inhalation led to reduced neutrophil migration into infected spaces and a higher burden of Pseudomonas aeruginosa. Taken together, these findings provide evidence that E‐cigarette use adversely impacts the innate immune system, potentially placing E‐cigarette users at higher risk for dysregulated inflammatory responses and invasive bacterial infections.

Interleukin-33 facilitates cutaneous defense against Staphylococcus aureus by promoting the development of neutrophil extracellular trap

Neutrophil extracellular traps (NETs) are consisted of DNA fibers and granular proteins and the formation of NETs has been identified as a crucial element of innate immune defense. IL-33 is a member of the IL-1 family cytokines and has been known as a strong trigger of type-2 immunity. Growing studies imply that IL-33 is involved in host defense against microbial infection. Here, we investigate the underlying influence of IL-33 on NET formation in mice with S. aureus cutaneous infection. We found that the level of IL-33 was significantly elevated in skin lesions of S. aureus-infected mice. The alarmin IL-33 inspired host innate defense through activation of NADPH oxidase to produce reactive oxygen species (ROS). Besides mediating the direct bactericidal activity within phagolysosomes, ROS production in IL-33-primed neutrophils was also critical for induction of NET formation. Enhancement of NET production by IL-33 contributed to ensnaring S. aureus and bacterial killing activity in vitro and in vivo. All together, these findings set up an IL-33/ST2 axis modulating NET generation, which strengthens host defense of innate immunity against S. aureus infection.

Differences in Staining for Neutrophil Elastase and its Controlling Inhibitor SLPI Reveal Heterogeneity among Neutrophils in Psoriasis

Neutrophils are broadly classified into conventional neutrophils (PMNs) and low-density granulocytes (LDGs). LDGs are better than PMNs at generating neutrophil extracellular traps (NETs), which may contribute to the pathology of autoimmune diseases. We hypothesized that LDGs and PMNs differ in their levels of unrestrained NE that supports NET generation. Here, we show that individuals with psoriasis contain elevated levels of LDGs and that in contrast to PMNs, the LDGs display higher staining for NE and lower staining for its inhibitor SLPI. The heterogeneity between blood-derived LDGs and PMNs was somewhat reminiscent of the differences in the NE and SLPI staining patterns observed in psoriasis skin-infiltrating neutrophils. Distinctive staining for NE and SLPI in LDGs and PMNs did not result from differences in their protein levels nor manifested in higher total proteolytic activity of NE in LDGs; rather, it likely depended on different cytosolic sequestration of these proteins. The disparate profile of NE and SLPI in LDGs and PMNs coincided with altered migratory responses of these cells to cutaneous chemoattractants. Collectively, differential NE and SLPI staining identifies common attributes of both circulating and skin-infiltrating neutrophils, which may guide neutrophil migration to distinct skin regions and determine the localization of LDGs-mediated cutaneous pathology.

Targeting neutrophil extracellular traps in severe acute pancreatitis treatment

Severe acute pancreatitis (SAP) is a critical abdominal disease associated with high death rates. A systemic inflammatory response promotes disease progression, resulting in multiple organ dysfunction. The functions of neutrophils in the pathology of SAP have been presumed traditionally to be activation of chemokine and cytokine cascades accompanying the inflammatory process. Recently, since their discovery, a new type of antimicrobial mechanism, neutrophil extracellular traps (NETs), and their role in SAP, has attracted widespread attention from the scientific community. Significantly different from phagocytosis and degranulation, NETs kill extracellular microorganisms by releasing DNA fibers decorated with granular proteins. In addition to their strong antimicrobial functions, NETs participate in the pathophysiological process of many noninfectious diseases. In SAP, NETs injure normal tissues under inflammatory stress, which is associated with the activation of inflammatory cells, to cause an inflammatory cascade, and SAP products also trigger NET formation. Thus, due to the interaction between NET generation and SAP, a treatment targeting NETs might become a key point in SAP therapy. In this review, we summarize the mechanism of NETs in protecting the host from pathogen invasion, the stimulus that triggers NET formation, organ injury associated with SAP involving NETs, methods to interrupt the harmful effects of NETs, and different therapeutic strategies to preserve the organ function of patients with SAP by targeting NETs.

Neutrophil extracellular traps (NETs) formation induced by TGF-β in oral lichen planus – Possible implications for the development of oral cancer

Oral Potentially Malignant Disorders (OPMDs) including Oral Lichen Planus (OLP) are associated with risk of transformation to oral squamous cell carcinoma (OSCC). Available data show that innate immune cells involving polymorphonuclear neutrophils (PMNs) with their ability to neutrophil extracellular traps (NETs) formation are likely to be directly involved in development of cancer. Examination of NETs generation by TGF-β - induced neutrophils of OLP patients showed increased amounts of traps with MPO, H3Cit and cfDNA, known to be released with NETs. The presence of excessive amounts of NETs components may lead to numerous adverse consequences associated with potential transformation to OSCC. Bacterial-related infection may enhance the NETs formation and lead to consequences resulting from the excessive number of individual elements of these networks. It is likely that regulating NETs release by the flavonoids presented herein may be beneficial not only for inhibiting OLP development, but also in reducing risk of transformation to OSCC.

MiR-155 Regulates PAD4-Dependent Formation of Neutrophil Extracellular Traps.

Accumulating data suggest that neutrophil extracellular traps (NETs) exert a key function in several diseases. Peptidylarginine deiminase 4 (PAD4) regulates NET formation via citrullination of histones. The aim of this study was to examine the role of miR-155 in controlling PAD4-dependent generation of NETs. Bone marrow neutrophils were stimulated with PMA and MIP-2. Pre-incubation of neutrophils with translational inhibitors (cycloheximide or puromycin) markedly decreased NET formation induced by PMA or MIP-2. Neutrophil transfection with a mimic miR-155 increased PMA-induced PAD4 mRNA expression and NET formation. In contrast, transfection with an antagomiR-155 decreased induction of PAD4 mRNA and NETs in response to PMA challenge. Bioinformatical examination of PAD4 revealed a potential binding site in AU-rich elements at the 3′-UTR region. MiR-155 binding to PAD4 was examined by use of target site blockers and RNA immunoprecipitation, revealing that miR-155 regulation of PAD4 mRNA is mediated via AU-rich elements in the 3′-UTR region. In conclusion, our findings demonstrate that miR-155 positively regulates neutrophil expression of PAD4 and expulsion of extracellular traps. Thus, our novel results indicate that targeting miR-155 might be useful to inhibit exaggerated NET generation in inflammatory diseases.

Activation leads to a significant shift in the intracellular redox homeostasis of neutrophil-like cells

Neutrophils produce a cocktail of oxidative species during the so-called oxidative burst to attack phagocytized bacteria. However, little is known about the neutrophils' redox homeostasis during the oxidative burst and there is currently no consensus about the interplay between oxidative species and cellular signaling, e.g. during the initiation of the production of neutrophil extracellular traps (NETs). Using the genetically encoded redox sensor roGFP2, expressed in the cytoplasm of the neutrophil-like cell line PLB-985, we saw that stimulation by both PMA and E. coli resulted in oxidation of the thiol residues in this probe. In contrast to the redox state of phagocytized bacteria, which completely breaks down, the neutrophils' cytoplasmic redox state switched from its intital -318 ± 6 mV to a new, albeit higher oxidized, steady state of -264 ± 5 mV in the presence of bacteria. This highly significant oxidation of the cytosol (p value = 7 × 10-5) is dependent on NOX2 activity, but independent of the most effective thiol oxidant produced in neutrophils, MPO-derived HOCl. While the shift in the intracellular redox potential is correlated with effective NETosis, it is, by itself not sufficient: Inhibition of MPO, while not affecting the cytosolic oxidation, significantly decreased NETosis. Furthermore, inhibition of PI3K, which abrogates cytosolic oxidation, did not fully prevent NETosis induced by phagocytosis of bacteria. Thus, we conclude that NET-formation is regulated in a multifactorial way, in part by changes of the cytosolic thiol redox homeostasis in neutrophils, depending on the circumstance under which the generation of NETs was initiated.

Increased plasmatic NETs by-products in patients in severe obesity

Neutrophil extracellular traps (NETs) are DNAs products involved in immune process. Obesity through a low-grade chronic inflammation determines neutrophil activation, but it is still unclear its role in NETs formation. Here we analyzed the NETs levels in healthy and morbid obese, their association with anthropometric and glyco-metabolic parameters and their changes after bariatric surgery. For this study, we enrolled 73 patients with morbid obesity (BMI ≥40 kg/m2 or ≥35 kg/m2 + comorbidity) eligible to sleeve gastrectomy. In parallel, 55 healthy subjects and 21 patients with severe coronary artery disease were studied as controls. We evaluated anthropometric parameters, peripheral blood pressure, biochemical and serum analysis at the enrollment and at twelve months after surgery. Plasmatic levels of MPO-DNA complexes were assessed by ELISA. NETs levels were higher in obese than in control group (p < 0.001) and correlated with the main anthropometric variable (BMI, waist, hip), glyco-metabolic variables and systolic blood pressure. NETs trend after intervention was uneven. The reduction of NETs correlated with the entity of reduction of BMI (ρ = 0.416, p < 0.05), visceral fat area (ρ = 0.351, p < 0.05), and glycemia (ρ = 0.495, p < 0.001). In medical history of patients in whom NETs increased, we observed a higher number of thromboembolic events. Our observations indicate that severe obesity is associated with increased generation of NETs, which in turn could influence the patients’ systemic inflammatory state. Weight loss and in particular, loss of adipose tissue after bariatric surgery does not in itself correct NET’s dysregulated production. Finally, patients in whom NETs accumulation persists after surgery are probably those at the highest risk of cardiovascular events.

IL-1β Blockade Attenuates Thrombosis in a Neutrophil Extracellular Trap-Dependent Breast Cancer Model.

Cancer patients are at increased risk of developing thrombosis, comorbidity that has been associated with increased neutrophil counts and the formation of neutrophil extracellular traps (NETs). Interleukin-1β (IL-1β) modulates the expression of granulocyte colony-stimulating factor (G-CSF), a cytokine that promotes cancer-associated neutrophilia and NET generation. Herein, we combined a murine breast cancer model with a flow-restriction thrombosis model to evaluate whether the IL-1β blockade could interfere with cancer-associated thrombosis. Mice bearing metastatic 4T1 tumors exhibited high neutrophil counts as well as elevated expression of G-CSF and IL-1β in their tumors. On the other hand, mice bearing non-metastatic 67NR tumors showed no elevation in neutrophil counts and displayed low expression levels of G-CSF and IL-1β in their tumors. 4T1 tumor-bearing mice but not 67NR tumor-bearing mice exhibited a NET-dependent prothrombotic state. Pharmacological blockade of IL-1 receptor (IL-1R) decreased the primary growth of 4T1 tumors and reduced the systemic levels of myeloperoxidase, cell-free DNA (cfDNA) and G-CSF, without interfering with the neutrophil counts. Most remarkably, the blockade of IL-1R abolished the prothrombotic state observed in 4T1 tumor-bearing mice. Overall, our results demonstrate that IL-1β might be a feasible target to attenuate cancer-associated thrombosis, particularly in cancer types that rely on increased G-CSF production and involvement of NET formation.

Abstract P1126: Neutrophil Extracellular Traps: New Players in Hypertension

Aberrant immune responses are linked to hypertension; yet, neutrophils remain relatively understudied. Both neutrophilia and elevated neutrophil-to-lymphocyte (N/L) ratio are used as clinical biomarkers of hypertension. Likewise, neutrophilia is reported in spontaneously hypertensive rats. Whether these features are associated with an increase in neutrophil extracellular traps (NETs) generation is unknown. We hypothesized that neutrophilia and NETs directly correlate with hypertension whereby lowering neutrophilia and NETs are therapeutically beneficial to lower blood pressure (BP). Herein, we used various, genetic rat models divergent in BP to study the neutrophil-hypertension axis. These include the Dahl Salt-Sensitive (S) and Resistant (R) rats, and the low (LCR) and high (HCR) exercise capacity runner rats. LCR (2.1±0.3 10 9 /l) and S rats (1.2±0.5 10 9 /l) exhibited neutrophilia compared to HCR (0.98±0.5 10 9 /l) and R rats (1.04±0.5 10 9 /l), p&lt;0.05, respectively. To determine whether neutrophil function was also augmented, peripheral neutrophils were stimulated with either PMA or LPS to induce ROS and NETs generation, respectively. LCR and S rats generated ~2-3 folds more ROS and NETs compared to their HCR and R counterparts. Intriguingly, LCR (11.4±1.0 U/ml) displayed reduced DNase I activity than HCR (17.8±0.8 U/ml), p&lt;0.05, which indicated an inadequate clearance of NETs. Systemic and renal lipocalin-2 were 2-3 folds higher in the S rats compared to R rats, suggesting low-grade inflammation associated with NETs overabundance. Introducing a high-salt diet to S and R rats further potentiated neutrophilia, ROS and NETs, the extent of elevation being higher in S rats. Opposingly, oral administration of β-hydroxybutyrate (βOHB; a hypotensive ketone body) to S and R rats reduced neutrophilia, NETs and ROS. Similarly, subjecting LCR to exercise and fasting, both of which lower BP and increase βOHB, substantially attenuated neutrophilia, NETs and ROS. Collectively, our results demonstrate that NETs are an important feature directly linked to elevated BP in a strain-independent manner. Thus, blunting neutrophil responses through dietary intervention or exercise could serve as a novel therapeutic strategy against hypertension.

Transketolase and vitamin B1 influence on ROS-dependent neutrophil extracellular traps (NETs) formation.

Neutrophil extracellular traps (NETs) are a recently identified, web-like, extracellular structure composed of decondensed nuclear DNA and associated antimicrobial granules. NETs are extruded into the extracellular environment via the reactive oxygen species (ROS)-dependent cell death pathway participating in inflammation and autoimmune diseases. Transketolase (TKT) is a thiamine pyrophosphate (vitamin B1)-dependent enzyme that links the pentose phosphate pathway with the glycolytic pathway by feeding excess sugar phosphates into the main carbohydrate metabolic pathways to generate biosynthetic reducing capacity in the form of NADPH as a substrate for ROS generation. In this work, TKT was selected as a lead candidate from 24 NET-associated proteins obtained by literature screening and knowledge gap assessment. Consequently, we determined whether TKT influenced NET formation in vitro. We firstly established that the release of ROS-dependent NETs was significantly decreased after purified human PMNs were pretreated with oxythiamine, a TKT inhibitor, and in a concentration dependent manner. As a cofactor for TKT reaction, we evaluated the release of NET formation either in vitamin B1 treatment or in combined use of oxythiamine and vitamin B1, and found that those treatments also exerted a significant suppressive effect on the amount of NET-DNA and ROS production. The regulation of TKT by oxythiamine and/or vitamin B1 may therefore be associated with response to the modulation of NET formation by preventing generation of excessive NETs in inflammatory diseases.

Extracellular DNA NET-Works With Dire Consequences for Health.

Neutrophils play a central role in innate immune defense. Advances in neutrophil biology have brought to light the capacity of neutrophils to release their decondensed chromatin and form large extracellular DNA networks called neutrophil extracellular traps (NETs). NETs are produced in response to many infectious and noninfectious stimuli and, together with fibrin, block the invasion of pathogens. However, their formation in inflamed blood vessels produces a scaffold that supports thrombosis, generates neo-antigens favoring autoimmunity, and aggravates damage in ischemia/reperfusion injury. NET formation can also be induced by cancer and promotes tumor progression. Formation of NETs within organs can be immediately detrimental, such as in lung alveoli, where they affect respiration, or they can be harmful over longer periods of time. For example, NETs initiate excessive deposition of collagen, resulting in fibrosis, thus likely contributing to heart failure. Here, we summarize the latest knowledge on NET generation and discuss how excessive NET formation mediates propagation of thrombosis and inflammation and, thereby, contributes to various diseases. There are many ways in which NET formation could be averted or NETs neutralized to prevent their detrimental consequences, and we will provide an overview of these possibilities.

NETs promote ALI/ARDS inflammation by regulating alveolar macrophage polarization

Neutrophils activated during acute lung injury (ALI) form neutrophil extracellular traps (NETs) to capture pathogens. However, excessive NETs can cause severe inflammatory reactions. Macrophages are classified as M1 macrophages with proinflammatory effects or M2 macrophages with anti-inflammatory effects. During ALI, alveolar macrophages (AMs) polarize to the M1 phenotype. This study tested the hypothesis that NETs may aggravate ALI or acute respiratory distress syndrome (ARDS) inflammation by promoting alveolar macrophage polarization to the M1 type. Our research was carried out in three aspects: clinical research, animal experiments and in vitro experiments. We determined that NET levels in ARDS patients were positively correlated with M1-like macrophage polarization. NET formation was detected in murine ALI tissue and associated with increased M1 markers and decreased M2 markers in BALF and lung tissue. Treatment with NET inhibitors significantly inhibitor NETs generation, downregulated M1 markers and upregulated M2 markers. Regardless of LPS pre-stimulation, significant secretion of proinflammatory cytokines and upregulated M1 markers were detected from bone marrow-derived macrophages (M0 and M2) cocultured with high concentrations of NETs; conversely, M2 markers were downregulated. In conclusion, NETs promote ARDS inflammation during the acute phase by promoting macrophage polarization to the M1 phenotype. We propose that NETs play an important role in the interaction between neutrophils and macrophages during the early acute phase of ALI.

GnRH impairs diabetic wound healing through enhanced NETosis

It has been reported that neutrophil extracellular traps (NETs) impair wound healing in diabetes and that inhibiting NET generation (NETosis) improves wound healing in diabetic mice. Gonadotropin-releasing hormone (GnRH) agonists are associated with a greater risk of diabetes. However, the role of GnRH in diabetic wound healing is unclear. We determined whether GnRH-promoted NETosis and induced more severe and delayed diabetic wound healing. A mouse model of diabetes was established using five injections with streptozotocin. Mice with blood glucose levels >250 mg/dL were then used in the experiments. GnRH agonist treatment induced delayed wound healing and increased NETosis at the skin wounds of diabetic mice. In contrast, GnRH antagonist treatment inhibited GnRH agonist-induced delayed wound healing. The expression of NETosis markers PAD4 and citrullinated histone H3 were increased in the GnRH-treated diabetic skin wounds in diabetic mice and patients. In vitro experiments also showed that neutrophils expressed a GnRH receptor and that GnRH agonist treatment increased NETosis markers and promoted phorbol myristate acetate (PMA)-induced NETosis in mouse and human neutrophils. Furthermore, GnRH antagonist treatment suppressed the expression of NETosis markers and PMA-induced NETosis, which were increased by GnRH treatment. These results indicated that GnRH-promoted NETosis and that increased NETosis induced delayed wound healing in diabetic skin wounds. Thus, inhibition of GnRH might be a novel treatment of diabetic foot ulcers.

PS1497 NEUTROPHIL EXTRACELLULAR TRAPS (NETS), BLOOD CELL COUNTS AND THROMBOTIC RISK IN ITP PATIENTS

Background:Patients with immune thrombocytopenia (ITP) are at increased risk of vascular events (VE), but the precise mechanisms of this prothrombotic state remain largely unknown. Neutrophil extracellular traps (NETs) are DNA‐containing structures released by neutrophils that are increasingly being reported in patients with infection and thrombosis associated with various autoimmune and non‐immune disorders. However, the contribution of NETs in the prothrombotic state in ITP patients is largely unknown.Aims:To determine if ITP patients have enhanced intrinsic potential for NET formation and to evaluate their prothrombotic role in ITP, their correlation with platelet activation, and also with specific therapeutic approaches.Methods:Sixty‐three ITP patients at different stages of disease and 30 healthy controls were included. NETs were evaluated by quantifying cell free DNA (cfDNA) using SYTOX Green, and by assessing the levels of Citrullinated Histone H3 complexed to DNA (H3Cit‐DNA) via a sandwich ELISA. Platelet and white blood cell (WBC) counts were assessed by a cell counter (Sysmex), and platelet glycoproteins, size, and degranulation were evaluated by flow cytometry.Results:The levels of cfDNA and H3Cit‐DNA in the peripheral blood of ITP patients were higher than in controls (p = 0.006, and p = 0.001, respectively). The percentage of patients with detectable H3Cit‐DNA complexes (optical density &gt;0.199) was significantly different to that of controls (26.2% vs. 6.7%; p = 0.029). In patients, positivity for H3Cit‐DNA correlated with higher cfDNA (Pearson correlation: R = 0.407; p = 0.01). No significant differences in the levels of cfDNA or H3Cit‐DNA were detected between the patients in remission (n = 24) and those with active ITP (n = 39). Seven ITP patients (11.1%) had experienced previous VE and presented with increased cfDNA (0.207 vs. 0.152 ng/ml; p = 0.034), WBC count (9.6 vs. 8.0 x109/L; p = 0.037), and neutrophil count (5.4 vs. 4.3x109/L; p = 0.027) compared to patients with no history of thrombotic events. Only one of the patients with VE was under low dose prednisone. Previous thrombosis was neither associated to H3Cit‐cfDNA positivity, platelet counts, mean fluorescence intensity of platelet glycoprotein expression (GPIba, aIIb), size (FSC), nor percentage of degranulated platelets (CD62, CD63). When we evaluated whether other acquired risk factors correlated with cfDNA, neither previous splenectomy, hypertension, hypercholesterolemia, smoking, age, nor diabetes correlated with cfDNA. Notably, treatment with thrombopoietin receptor agonists, corticosteroids, or immunosuppresants at sampling were neither related to circulating NETs cfDNA or H3Cit‐DNA) nor to platelet activation.Summary/Conclusion:Our results show that plasma NET levels are elevated in ITP patients, and that this increase is especially marked in patients who have suffered from a vascular event. The fact that patients with previous thrombosis had increased WBC and neutrophil counts suggests that there may be more NETosis in these individuals, probably indicating a dysregulation between production and clearance. Other factors (platelet activation, acquired thrombotic risk factors or current therapy) did not seem to influence NET formation or platelet degradation. Similar to patients with cancer and myeloproliferative disorders, where leukocytosis has been associated with increased thromboembolic risk, we propose an association between NET generation and neutrophil counts leading to increased risk of thrombosis in ITP, which may provide new therapeutic targets to combat VE in ITP.Funding: ISCIII and FEDER [PI17/00051].

Novel findings in neutrophil biology and their impact on cardiovascular disease.

Neutrophils are the most abundant circulating leucocytes in healthy humans. These cells are central players during acute inflammatory responses, although a growing body of evidence supports a crucial role in chronic inflammation and chemokines and cytokines related to it as well. Thus, both humoral and cellular components are involved in the development of plaque formation and atherosclerosis. Accordingly, CANTOS trial using an interleukin-1 beta antibody confirmed that inflammatory cytokines contribute to the occurrence of myocardial infarction and cardiac death independent of changes in lipids. Recent data revealed that neutrophils are a heterogeneous population with different subsets and functional characteristics (i.e. CD177+ cells, OLFM4+ neutrophils, proangiogenic neutrophils, neutrophils undergoing reverse migration, and aged neutrophils). Importantly, neutrophils are able to synthesize de novo proteins. Neutrophil extracellular trap generation and NETosis have been considered as very important weapons in sterile inflammation. Neutrophil-derived microvesicles represent another mechanism by which neutrophils amplify inflammatory processes, being found at high levels both at the site of injury and in the bloodstream. Finally, neutrophil aging can influence their functions also in relation with host age. These recent acquisitions in the field of neutrophil biology might pave the way for new therapeutic targets to prevent or even treat patients experiencing cardiovascular (CV) diseases. Here, we discuss novel findings in neutrophil biology, their impact on CV and cerebrovascular diseases, and the potential implementation of these notions into daily clinical practice.

Stimulation of the class-A scavenger receptor induces neutrophil extracellular traps (NETs) by ERK dependent NOX2 and ROMO1 activation

Neutrophil extracellular traps (NETs) play a critical role in host antimicrobial response whereas they are also implicated in the pathogenesis of inflammatory and autoimmunediseases. Generation of reactiveoxygen species (ROS) is key to NETs formation. A variety of stimulatory ligands have been found to enhance ROS production and thus trigger NETs. However, the mechanisms that connect receptor stimuli with ROS production and NETs formation remain unclear. In this study, we described a new mechanism of NETs generation in neutrophils triggered by stimulation of the class A scavenger receptor (SRA), a major subtype of scavenger receptors in response to various stimuli during infection and inflammatory disorders. By using polyinosinic acid (Poly I), a ribonucleotide ligand of SRA, we demonstrated that SRA stimulation lead to selective ERK phosphorylation, which upregulated cytosol ROS levels and induced canonical NETs formation by activating NADPH oxidase 2 (NOX2). Interestingly, our results showed that mitochondrial ROS (mtROS) production was also enhanced by the SRA dependent ERK activation through upregulation and activation of reactive oxygen species modulator 1(ROMO1), a mitochondrial membrane protein and a key mediator of mtROS. Moreover, inhibition of the SRA elicited ROMO1 activation dampened NETs release upon SRA stimulation. Overall, our study describes a new insight into the NETs release triggered by membrane SRA stimulation and mediated by ERK dependent NOX2 and ROMO1 activation.

P153 LOSS OF NEUTROPHIL EXTRACELLULAR TRAPS VIA PEPTIDYL ARGININE DEIMINASE-4 DEFICIENCY AGGRAVATES CITROBACTER RODENTIUM- INDUCED GUT INFLAMMATION

Citrobacter rodentium (CR) is an attaching-effacing enteropathogen which infects mice and causes intestinal inflammation. The CR infection model is appropriate for investigating host-pathogen interactions in the gut and understanding the pathogenesis of human inflammatory bowel diseases. Peptidyl arginine deiminase-4 (PAD4) is an enzyme required for the generation of neutrophil extracellular traps (NETs), which can provide antimicrobial effects during the host innate immune response; however, the role of PAD4 against gastrointestinal (GI) infection is largely unknown. In this study, we challenged PAD4-deficient (Pad4-/-) mice and wild type (WT) littermates against CR, and investigated bacteria clearance and gut pathology. Luminal colonization of CR in Pad4-/- mice peaked between 11-14 days post-infection (p.i.), whereas WT mice suppressed the infection by 14 days. Loss of PAD4 substantially increased CR dissemination to the spleen and mesenteric lymph nodes, which prolonged and aggravated CR-associated inflammation as indicated by splenomegaly, colomegaly and elevated systemic and colonic pro-inflammatory markers (lipocalin 2 and serum amyloid A). Likewise, the colonic antimicrobial cytokines, IL-22 and IL-17A, were more upregulated in Pad4-/- than WT mice in response to infection. Histological analysis revealed that transmissible colonic hyperplasia and goblet cell depletion were more pronounced in Pad4-/- than WT mice. Using a bioluminescent strain of CR (ICC180), we observed that the transit of CR through the gastrointestinal tract occurs more rapidly in Pad4-/- mice. Treating WT mice with deoxyribonuclease I (DNase I; disrupts NETs generation), recapitulated the exacerbated CR infection and gut pathology as in PAD4 deficient mice. Administration of the PAD4 inhibitor, Cl-amidine, also aggravated CR infection, but to a lesser extent than those observed in either Pad4-/- or DNase I-treated mice. Taken together, our findings highlight the importance of PAD4 in the mucosal clearance of CR and in resolving the gut-associated inflammation and colitis. In light of the ongoing efforts to target PAD4 or NETs to treat other inflammatory disorders, our findings caution that their inhibition may not be favorable against GI infection.