Serine proteases in neutrophil extracellular traps exhibit anti-Respiratory Syncytial Virus activity.

Human respiratory syncytial virus (hRSV) is an infectious agent in infants and young children which there are no vaccines or drugs for treatment. Neutrophils are recruited for airway, where they are stimulated by hRSV to release large amounts of neutrophil extracellular traps (NETs). NETs are compound by DNA and proteins, including microbicidal enzymes. They constitute a large part of the mucus accumulated in the lung of patients, compromising their breathing capacity. In contrast, NETs can capture/inactivate hRSV, but the molecules responsible for this effect are unknown. OBJECTIVES: We selected microbicidal NET enzymes (elastase, myeloperoxidase, cathepsin-G, and proteinase-3) to assess their anti-hRSV role. METHODS AND RESULTS: Through in vitro assays using HEp-2 cells, we observed that elastase, proteinase-3, and cathepsin-G, but not myeloperoxidase, showed virucidal effects even at non-cytotoxic concentrations. Elastase and proteinase-3, but not cathepsin-G, cleaved viral F-protein, which is responsible for viral adhesion and fusion with the target cells. Molecular docking analysis indicated the interaction of these macromolecules in the antigenic regions of F-protein through the active regions of the enzymes. CONCLUSIONS: Serine proteases from NETs interact and inactive hRSV. These results contribute to the understanding the role of NETs in hRSV infection and to designing treatment strategies for the inflammatory process during respiratory infections.

Generation of neutrophil extracellular traps in patients with acute liver failure is associated with poor outcome.

BACKGROUND AND AIMS: Acute liver failure (ALF) is characterized by significant changes in the hemostatic system and by systemic inflammation. The formation of neutrophil extracellular traps (NETs), in which an activated neutrophil expels its DNA, histones, and granular enzymes, such as myeloperoxidase (MPO), has been associated with immune-mediated and thrombotic diseases. We hypothesized that formation of NETs in patients with ALF contributes to progression of disease. APPROACH AND RESULTS: A total of 676 patients with ALF (international normalized ratio [INR], ≥1.5) or severe acute liver injury (ALI; INR, ≥2.0) were recruited from the U.S. ALF Study Group Registry between 2011 and 2018, of whom 308 patients (45.6%) had acetaminophen-induced ALF. Up to 21 days after admission, 483 patients (71.5%) survived without liver transplantation (LT). Levels of cell-free DNA (cfDNA) and the specific NET marker MPO-DNA complexes were measured in plasma samples obtained on admission and compared to levels in healthy controls. In addition, liver tissue obtained at transplantation of 20 ALF patients was stained for NETs. Levels of cfDNA were 7.1-fold, and MPO-DNA complexes 2.5-fold, higher in patients with ALF compared to healthy controls. cfDNA levels were not associated with 21-day transplant-free survival, but were higher in those patients with more-severe disease on admission, as reflected by various laboratory and clinical parameters. MPO-DNA levels were 30% higher in patients with ALF who died or required urgent LT. Liver tissue of ALF patients stained positive for NETs in 12 of 18 evaluable patients. CONCLUSIONS: Here, we provide evidence for NET formation in patients with ALF. Elevated plasma levels of MPO-DNA complexes in patients with ALF were associated with poor outcome, which suggests that NET formation contributes to disease progression.

Muscle Derived Mesenchymal Stem Cells Inhibit the Activity of the Free and the Neutrophil Extracellular Trap (NET)-Bond Myeloperoxidase.

Mesenchymal stem cells (MSCs) are known to migrate to tissue injury sites to participate in immune modulation, tissue remodelling and wound healing, reducing tissue damage. Upon neutrophil activation, there is a release of myeloperoxidase (MPO), an oxidant enzyme. But little is known about the direct role of MSCs on MPO activity. The aim of this study was to investigate the effect of equine mesenchymal stem cells derived from muscle microinvasive biopsy (mdMSC) on the oxidant response of neutrophils and particularly on the activity of the myeloperoxidase released by stimulated equine neutrophils. After specific treatment (trypsin and washings in phosphate buffer saline), the mdMSCs were exposed to isolated neutrophils. The effect of the suspended mdMSCs was studied on the ROS production and the release of total and active MPO by stimulated neutrophils and specifically on the activity of MPO in a neutrophil-free model. Additionally, we developed a model combining adherent mdMSCs with neutrophils to study total and active MPO from the neutrophil extracellular trap (NET). Our results show that mdMSCs inhibited the ROS production, the activity of MPO released by stimulated neutrophils and the activity of MPO bound to the NET. Moreover, the co-incubation of mdMSCs directly with MPO results in a strong inhibition of the peroxidase activity of MPO, probably by affecting the active site of the enzyme. We confirm the strong potential of mdMSCs to lower the oxidant response of neutrophils. The novelty of our study is an evident inhibition of the activity of MPO by MSCs. The results indicated a new potential therapeutic approach of mdMSCs in the inhibition of MPO, which is considered as a pro-oxidant actor in numerous chronic and acute inflammatory pathologies.

Interplay between Extracellular Matrix and Neutrophils in Diseases.

The extracellular matrix (ECM) is a highly dynamic and complex network structure, which exists in almost all tissues and is the microenvironment that cells rely on for survival. ECM interacts with cells to regulate diverse functions, including differentiation, proliferation, and migration. Neutrophils are the most abundant immune cells in circulation and play key roles in orchestrating a complex series of events during inflammation. Neutrophils can also mediate ECM remodeling by providing specific matrix-remodeling enzymes (such as neutrophil elastase and metalloproteinases), generating neutrophil extracellular traps, and releasing exosomes. In turn, ECM can remodel the inflammatory microenvironment by regulating the function of neutrophils, which drives disease progression. Both the presence of ECM and the interplay between neutrophils and their extracellular matrices are considered an important and outstanding mechanistic aspect of inflammation. In this review, the importance of ECM will be considered, together with the discussion of recent advances in understanding the underlying mechanisms of the intricate interplay between ECM and neutrophils. A better comprehension of immune cell-matrix reciprocal dependence has exciting implications for the development of new therapeutic options for neutrophil-associated infectious and inflammatory diseases.

Antibodies Against Pseudomonas aeruginosa Alkaline Protease Directly Enhance Disruption of Neutrophil Extracellular Traps Mediated by This Enzyme.

Extracellular traps released by neutrophils (NETs) are essential for the clearance of Pseudomonas aeruginosa. Alkaline protease (AprA) secreted by P. aeruginosa negatively correlates with clinical improvement. Moreover, anti-AprA in patients with cystic fibrosis (CF) can help identify patients with aggressive forms of chronic infection. However, the mechanism underlying the clinical outcomes remains unclear. We demonstrated that aprA deficiency in P. aeruginosa decreased the bacterial burden and reduced lung infection. AprA degraded NET components in vitro and in vivo but did not affect NET formation. Importantly, antibodies induced by AprA acted as an agonist and directly enhanced the degrading activities of AprA. Moreover, antisera from patients with P. aeruginosa infection exhibited antibody-dependent enhancement (ADE) similar to that of the antibodies we prepared. Our further investigations showed that the interaction between AprA and the specific antibodies might make the enzyme active sites better exposed, and subsequently enhance the recognition of substrates and accelerate the degradation. Our findings revealed that AprA secreted by P. aeruginosa may aggravate infection by destroying formed NETs, an effect that was further enhanced by its antibodies.

Peptidylarginine Deiminase 4 as a Possible Biomarker of Plaque Instability in Carotid Artery Stenosis.

BACKGROUND AND PURPOSE: Neutrophil extracellular traps (NETs) exhibit pro-inflammatory and pro-thrombotic properties. However, they have only been reported as important regulators in atherosclerosis, especially in atherothrombosis. We investigated the presence of NETs and plaque instability in patients with carotid artery stenosis. MATERIAL AND METHODS: A total of 39 consecutive patients with carotid artery stenosis were evaluated. All patients underwent carotid artery stenting (CAS) with dual protection (simultaneous flow reversal + distal filter) and blood aspiration as a method of distal embolism prevention. Local arterial blood was aspirated at the stent site and peptidylarginine deiminase 4 (PAD4), which is essential for the formation of NETs, was measured. The relationships between PAD4 and the patient profile, blood examination and plaque data were investigated. RESULTS: The mean value of PAD4 in local arterial blood in CAS was 0.5 ng/ml. Bivariate analysis demonstrated that PAD4 was associated with the neutrophil to lymphocyte ratio (p = 0.007), high-density lipoprotein (p = 0.02), triglycerides to high-density lipoprotein ratio (p = 0.007), ulceration (p = 0.02) and plaque contrast enhancement on T1 black blood imaging (p = 0.03). In multiple linear regression analyses, PAD4 was correlated with the neutrophil to lymphocyte ratio (p = 0.01) and ulceration (p = 0.01, cut-off value: 0.49 odds ratio: 19.3). CONCLUSIONS: PAD4, representative of the presence of NETs, was high in carotid plaques with unstable features. The neutrophil to lymphocyte ratio in peripheral blood was suggested to be a biomarker of vulnerable plaques. Elucidating the role of NETs may aid in clarifying factors that promote the instability of carotid plaques.

A Novel Peptidylarginine Deiminase 4 (PAD4) Inhibitor BMS-P5 Blocks Formation of Neutrophil Extracellular Traps and Delays Progression of Multiple Myeloma.

Multiple myeloma is a plasma cell malignancy, which grows in the bone marrow (BM). The major population of cells in the BM is represented by neutrophils and they can form neutrophil extracellular traps (NET). Here, we investigated whether multiple myeloma cells induce NET formation and whether targeting this process would delay multiple myeloma progression. We demonstrated that murine and human multiple myeloma cells stimulate citrullination of histone H3 and NET formation by neutrophils and that this process is abrogated by pharmacological targeting of peptidylarginine deiminase 4 (PAD4) with a novel-specific small molecule inhibitor BMS-P5. Administration of BMS-P5 to multiple myeloma-bearing mice delays appearance of symptoms and disease progression. Taken together, our data demonstrate that targeting PAD4 may be beneficial for treatment of multiple myeloma.

DNA-bound elastase of neutrophil extracellular traps degrades plasminogen, reduces plasmin formation, and decreases fibrinolysis: proof of concept in septic shock plasma.

Activation of platelets and neutrophils in septic shock results in the formation of microvascular clots containing an intricate scaffold of fibrin with neutrophil extracellular traps (NETs) DNA. NETs contain multiple components that might impact endogenous fibrinolysis, resulting in failure to lyse clots in the microcirculation and residual systemic microthrombosis. We propose herein that the reservoir of human neutrophil elastase (HNE) on NETs may directly interfere with the fibrinolytic mechanism via a plasminogen proteolytic pathway. To investigate this mechanism, we constructed fibrin-NETs matrices by seeding and activating neutrophils onto a fibrin surface and monitored plasminogen activation or degradation. We demonstrate that the elastase activity of HNE-DNA complexes is protected from inhibition by plasma antiproteases and sustains its ability to degrade plasminogen. Using mass spectrometry proteomic analysis, we identified plasminogen fragments composed of kringle (K) domains (K1+2+3, k1+2+3+4) and the serine protease (SP) region (K5-SP). We further demonstrate that patients with septic shock with disseminated intravascular coagulation have circulating HNE-DNA complexes, HNE-derived plasminogen fragments, a low plasminogen concentration, and a reduced capacity to generate plasmin onto fibrin. In conclusion, we show that NETs bearing active HNE-DNA complexes reduce plasminogen into fragments, thus impairing fibrinolysis by decreasing the local plasminogen concentration, plasminogen binding to fibrin, and localized plasmin formation.-Barbosa da Cruz, D., Helms, J., Aquino, L. R., Stiel, L., Cougourdan, L., Broussard, C., Chafey, P., Riès-Kautt, M., Meziani, F., Toti, F., Gaussem, P., Anglés-Cano, E. DNA-bound elastase of neutrophil extracellular traps degrades plasminogen, reduces plasmin formation, and decreases fibrinolysis: proof of concept in septic shock plasma.

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.

Impairment of the antifibrotic prostaglandin E2 pathway may influence neutrophil extracellular traps-induced fibrosis in the mare endometrium.

Prostaglandin E2 (PGE2) has contradictory effects in many organs. It may have proinflammatory, anti-inflammatory, or anti-fibrotic roles, depending on the type of receptors to which it binds. By signaling through its receptors EP2 and EP4, PGE2 mediates anti-inflammatory and anti-fibrotic actions. In spite of chronic endometrial fibrosis (endometrosis) being a major cause of mare infertility, its pathogenesis is not fully understood. We have shown that contact of mare endometrium in vitro with neutrophil extracellular traps (NETs) proteases favors endometrial collagen type I production. Therefore, we investigated the involvement of the PGE2 pathway in collagen deposition in mare endometrium, challenged in vitro with proteases present in NETs. Mare endometria (Kenney and Doig categories I/IIA and IIB/III), obtained in the follicular phase (FLP) and mid-luteal phase (MLP), were incubated for 24 h with components found in NETs (elastase, cathepsin-G, and myeloperoxidase). Secretion of PGE2 and transcripts for specific PGE synthase (PGES) and PGE2 receptors (EP2 and EP4) were evaluated. Impaired PGE2 production and low EP2 transcript abundance depended on the endometrial category and estrous cycle phase. Impairment of PGE2 and/or EP2 might play a role in FLP (category IIB/III) and MLP (I/IIA) endometrial fibrogenesis because of the reduction in its antifibrotic capacity. In conclusion, priming of the endometrium with endogenous ovarian steroids might inhibit the antifibrotic PGE2 pathway either in healthy or pathologic tissues with collagen formation after NETs proteases action.

Periodontal sources of citrullinated antigens and TLR agonists related to RA.

Anti-citrullinated protein autoantibodies (ACPA) precede the onset of clinical and subclinical rheumatoid arthritis (RA). ACPA are frequently generated in further chronic inflammatory diseases, e.g. chronic obstructive pulmonary disease, lupus, periodontitis (PD), characterized by citrullination and mucosal as well as systemic autoimmunity against citrullinated proteins. PD is of particular interest, as it exhibits two sources of citrullination, namely peptidylarginine deiminase 4 (PAD4) of periodontal neutrophils and neutrophil extracellular traps (NETs) as well as the PAD of Porphyromonas gingivalis (PPAD). Whereas the PAD4-citrullinated host peptides and/or proteins occur physiologically, PPAD-citrullinated ones appear under pathological conditions as neo-antigens. Frequently, the oral pathogens P. gingivalis and A. actinomycetemcomitans directly and indirectly participate in synovitis in RA, providing topical citrullination: P. gingivalis via PPAD and A. actinomycetemcomitans via leukotoxin A-mediated ROS-independent NET formation. In addition, transient bacteraemia due to tooth brushing indicates the possibility that citrullinated peptides and/or proteins from periodontium regularly enter the blood circulation. In this way, the mucosal firewall is evaded and the systemic immune response against citrullinated peptides and/or proteins is facilitated. However, the role of swallowed PD-derived sludge for the induction of oral tolerance remains to be established. We hypothesize (I) PD-driven endotoxemia may increase the host responsiveness to autoantigens via TLR4 activation and (II) this participates in development and propagation of RA (III) circulating PD-derived bacterial DNA is taken up by phagocytes, activates TLR9, and thus increases the responsiveness to autoantigens.

Neutrophil-released enzymes can influence composition of circulating immune complexes in multiple sclerosis.

During NET formation, the content of neutrophils granules is released into the intercellular milieu. Consisting of many proteases and ROS species, formed NETs were shown to degrade cytokines (Schauer, Nat Med, 2014); while the content of neutrophil's azurophilic granules proved to contain glycosidases, secreted upon activation (Thaysen-Andersen, JBC, 2015), and formation of autoantibodies to neutrophil beta-glucoronidase was connected with the level of anti-MPO antibodies (Ab) (Martensson, Autoimmunity, 1992). Taking into account these facts, we aimed to investigate the possibility of NET-related changes in glycan composition on circulating IgG molecules and IgG-IgM immune complexes in multiple sclerosis (MS). This autoimmune disorder still has no reliable detection markers or established ways of treatment, besides widely accepted interferon therapy, making it a particularly interesting clinical condition. By applying capture lectin-ELISA, we analysed binding of α2,6 sialyl-specific lectins SNA, PSqL, and core α1,6-fucose specific lectin AAL to circulating IgG and related complexes in five groups of MS patients: untreated (17 persons); undergoing therapy with interferon (IFN) ß-1 b (15 persons), corticosteroids (methylprednisolone) (12 persons) and anti-B-cell monoclonal Ab (12 persons: Ocrelizumab, 6 persons and alemtuzumab, 6 persons). A group of 23 healthy donors served as control. Significant increase in neutrophil elastase activity, observed in the group of patients under corticosteroid treatment was also accompanied by sialyl-specific PSqL and SNA lectin binding to captured IgG molecules. Subsequent analysis demonstrated that sialic acid residues were exposed on free IgG and on circulating IgG-IgM immune complexes. Increased lectin binding was not observed for anti-myelin basic protein (one of the major autoAb in MS) Ab compared to total serum Ab. IFN therapy was accompanied by low neutrophil elastase activity and low amount of circulating immune complexes. Incubation of in vitro generated NETs with human serum revealed the digestion of high-molecular weight immune complexes with subsequent exposure of hidden glycoepitops. Obtained data indicate the potential of neutrophil-derived proteases to modify (partially degrade) circulating immune complexes leading to exposure of internal glycoepitops.

Neutrophil extracellular traps produced during inflammation awaken dormant cancer cells in mice.

Cancer cells from a primary tumor can disseminate to other tissues, remaining dormant and clinically undetectable for many years. Little is known about the cues that cause these dormant cells to awaken, resume proliferating, and develop into metastases. Studying mouse models, we found that sustained lung inflammation caused by tobacco smoke exposure or nasal instillation of lipopolysaccharide converted disseminated, dormant cancer cells to aggressively growing metastases. Sustained inflammation induced the formation of neutrophil extracellular traps (NETs), and these were required for awakening dormant cancer. Mechanistic analysis revealed that two NET-associated proteases, neutrophil elastase and matrix metalloproteinase 9, sequentially cleaved laminin. The proteolytically remodeled laminin induced proliferation of dormant cancer cells by activating integrin α3ß1 signaling. Antibodies against NET-remodeled laminin prevented awakening of dormant cells. Therapies aimed at preventing dormant cell awakening could potentially prolong the survival of cancer patients.

Cleaved N-terminal histone tails distinguish between NADPH oxidase (NOX)-dependent and NOX-independent pathways of neutrophil extracellular trap formation.

OBJECTIVES: Neutrophil extracellular traps (NETs) act in various rheumatic diseases. Although NET formation was originally described as a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX)-dependent pathway, it appears that there are also NOX-independent pathways of NET release. Currently, no tools are available that can discriminate between both NET-forming pathways. We aimed to develop a serological method allowing the discrimination between NETs generated through NOX-dependent or NOX-independent pathways. METHODS: Histones from in vitro generated NOX-dependent and NOX-independent NETs were characterised with a panel of lupus-derived antibodies against N-terminal histone tails using immunofluorescence microscopy, western blot and ELISA. NETs in patients with NET-associated diseases, that is, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), psoriatic arthritis (PsA) and sepsis, were characterised in sandwich ELISAs employing antibodies against myeloperoxidase (MPO) and N-terminal histone tails as detecting and capturing antibodies, respectively. Functional responses of endothelial cells to NOX-dependent and NOX-independent NETs were assessed as well. RESULTS: Neutrophil elastase cleaves the N-terminal tails of core histones during NOX-dependent, but not during NOX-independent NET formation. Consequently, the detection of MPO-histone complexes with antibodies against N-terminal histone tails allows discrimination between NETs formed through a NOX-dependent or NOX-independent manner. Characterisation of in vivo circulating NETs revealed the presence of NOX-independent NETs in RA, SLE and sepsis, but NOX-dependent NETs in PsA. NOX-independent NETs displayed an increased capacity to activate endothelial cells when compared with NOX-dependent NETs. CONCLUSIONS: These results indicate heterogeneity in NET-forming pathways in vivo and highlight the need for disease-specific strategies to prevent NET-mediated pathology.

Neutrophil Extracellular Traps Induce Endothelial Cell Activation and Tissue Factor Production Through Interleukin-1α and Cathepsin G.

Objective- Coronary artery thrombosis can occur in the absence of plaque rupture because of superficial erosion. Erosion-prone atheromata associate with more neutrophil extracellular traps (NETs) than lesions with stable or rupture-prone characteristics. The effects of NETs on endothelial cell (EC) inflammatory and thrombogenic properties remain unknown. We hypothesized that NETs alter EC functions related to erosion-associated thrombosis. Approach and Results- Exposure of human ECs to NETs increased VCAM-1 (vascular cell adhesion molecule 1) and ICAM-1 (intercellular adhesion molecule 1) mRNA and protein expression in a time- and concentration-dependent manner. THP-1 monocytoid cells and primary human monocytes bound more avidly to NET-treated human umbilical vein ECs than to unstimulated cells under flow. Treatment of human ECs with NETs augmented the expression of TF (tissue factor) mRNA, increased EC TF activity, and hastened clotting of recalcified plasma. Anti-TF-neutralizing antibody blocked NET-induced acceleration of clotting by ECs. NETs alone did not exhibit TF activity or acceleration of clotting in cell-free assays. Pretreatment of NETs with anti-interleukin (IL)-1α-neutralizing antibody or IL-1Ra (IL-1 receptor antagonist)-but not with anti-IL-1ß-neutralizing antibody or control IgG-blocked NET-induced VCAM-1, ICAM-1, and TF expression. Inhibition of cathepsin G, a serine protease abundant in NETs, also limited the effect of NETs on EC activation. Cathepsin G potentiated the effect of IL-1α on ECs by cleaving the pro-IL-1α precursor and releasing the more potent mature IL-1α form. Conclusions- NETs promote EC activation and increased thrombogenicity through concerted action of IL-1α and cathepsin G. Thus, NETs may amplify and propagate EC dysfunction related to thrombosis because of superficial erosion.

Role of Peptidylarginine Deiminase 4 in Neutrophil Extracellular Trap Formation and Host Defense during Klebsiella pneumoniae-Induced Pneumonia-Derived Sepsis.

Peptidylarginine deiminase 4 (PAD4) catalyzes citrullination of histones, an important step for neutrophil extracellular trap (NET) formation. We aimed to determine the role of PAD4 during pneumonia. Markers of NET formation were measured in lavage fluid from airways of critically ill patients. NET formation and host defense were studied during pneumonia-derived sepsis caused by Klebsiella pneumoniae in PAD4+/+ and PAD4-/- mice. Patients with pneumosepsis, compared with those with nonpulmonary disease, showed increased citrullinated histone 3 (CitH3) levels in their airways and a trend toward elevated levels of NET markers cell-free DNA and nucleosomes. During murine pneumosepsis, CitH3 levels were increased in the lungs of PAD4+/+ but not of PAD4-/- mice. Combined light and electron microscopy showed NET-like structures surrounding Klebsiella in areas of CitH3 staining in the lung; however, these were also seen in PAD4-/- mice with absent CitH3 lung staining. Moreover, cell-free DNA and nucleosome levels were mostly similar in both groups. Moreover, Klebsiella and LPS could still induce NETosis in PAD4-/- neutrophils. Both groups showed largely similar bacterial growth, lung inflammation, and organ injury. In conclusion, these data argue against a major role for PAD4 in NET formation, host defense, or organ injury during pneumonia-derived sepsis.

Loss of Sirt3 accelerates arterial thrombosis by increasing formation of neutrophil extracellular traps and plasma tissue factor activity.

Aims: Sirtuin 3 (Sirt3) is a mitochondrial, nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase that reduces oxidative stress by activation of superoxide dismutase 2 (SOD2). Oxidative stress enhances arterial thrombosis. This study investigated the effects of genetic Sirt3 deletion on arterial thrombosis in mice in an inflammatory setting and assessed the clinical relevance of these findings in patients with ST-elevation myocardial infarction (STEMI). Methods and results: Using a laser-induced carotid thrombosis model with lipopolysaccharide (LPS) challenge, in vivo time to thrombotic occlusion in Sirt3-/- mice (n = 6) was reduced by half compared to Sirt3+/+ wild-type (n = 8, P < 0.01) controls. Ex vivo analyses of whole blood using rotational thromboelastometry revealed accelerated clot formation and increased clot stability in Sirt3-/- compared to wild-type blood. rotational thromboelastometry of cell-depleted plasma showed accelerated clotting initiation in Sirt3-/- mice, whereas overall clot formation and firmness remained unaffected. Ex vivo LPS-induced neutrophil extracellular trap formation was increased in Sirt3-/- bone marrow-derived neutrophils. Plasma tissue factor (TF) levels and activity were elevated in Sirt3-/- mice, whereas plasma levels of other coagulation factors and TF expression in arterial walls remained unchanged. SOD2 expression in bone marrow -derived Sirt3-/- neutrophils was reduced. In STEMI patients, transcriptional levels of Sirt3 and its target SOD2 were lower in CD14+ leukocytes compared with healthy donors (n = 10 each, P < 0.01). Conclusions: Sirt3 loss-of-function enhances experimental thrombosis in vivo via an increase of neutrophil extracellular traps and elevation of TF suggesting thrombo-protective effects of endogenous Sirt3. Acute coronary thrombosis in STEMI patients is associated with lower expression levels of SIRT3 and SOD2 in CD14+ leukocytes. Therefore, enhancing SIRT3 activity by pan-sirtuin activating NAD+-boosters may provide a novel therapeutic target to prevent or treat thrombotic arterial occlusion in myocardial infarction or stroke.

Myeloperoxidase influences the complement regulatory activity of complement factor H.

Objective: The interaction between neutrophils and activation of alternative complement pathway plays a critical role in the pathogenesis of ANCA-associated vasculitis (AAV). MPO, which can be released from ANCA-stimulated neutrophils, was recently demonstrated to be capable of activating the alternative complement pathway. Here we aimed to investigate the interaction between MPO and factor H (FH), a key regulator of the alternative pathway, and its effect on the functional activities of FH. Methods: Detection of FH and MPO on neutrophil extracellular traps (NETs) induced by serum from AAV patients and in kidney biopsies of AAV patients was performed by immunostaining. In vitro binding between MPO and FH was examined by ELISA and surface plasmon resonance. The influence of MPO on the complement regulatory activity of FH was further assessed. Results: FH deposited and co-localized with MPO in NETs. In kidney biopsies from AAV patients, MPO was closely adjacent to FH in glomerular capillaries. We demonstrated that MPO binds to FH with an apparent nanomolar affinity and identified short consensus repeats 1-4 of FH as the major binding sites. In terms of functional analysis, MPO inhibited the interaction between FH and C3b and the decay-accelerating activity of FH. The fluid phase and surface cofactor activities of FH upon C3b inactivation were inhibited by MPO. Conclusion: Our findings indicate that MPO binds to FH and influences the complement regulatory activity of FH. MPO-FH interaction may participate in the pathogenesis of AAV by contributing to activation of the alternative complement pathway.

Neutrophil peptidyl arginine deiminase-4 has a pivotal role in ischemia/reperfusion-induced acute kidney injury.

Ischemia/reperfusion is a common cause of acute kidney injury (AKI). However, mechanisms underlying the sudden loss in kidney function and tissue injury remain to be fully elucidated. Here, we investigated the role of peptidyl arginine deiminase-4 (PAD4), which converts arginine to citrulline and plays a role in epigenetic regulation and inflammation, in renal ischemia/reperfusion injury. PAD4 expression was highly induced in infiltrating leukocytes 24 hours following renal ischemia and reperfusion. This induction was accompanied by citrullination of histone H3 and formation of neutrophil extracellular traps in kidneys of wild-type mice. By contrast, PAD4-deficient mice did not form neutrophil extracellular traps, expressed lower levels of pro-inflammatory cytokines and were partially protected from renal ischemia/reperfusion-induced AKI. Furthermore, PAD4-deficient mice recovered kidney function 48 hours after ischemia/reperfusion, whereas kidney function in the wild-type mice progressively worsened. Administration of DNase I, which degrades neutrophil extracellular traps or the PAD-specific inhibitor YW3-56 before ischemia, partially prevented renal ischemia/reperfusion-induced AKI. Notably, transfer of neutrophils from wild-type, but not from PAD4-deficient mice, was sufficient to restore renal neutrophil extracellular trap formation and impair kidney function following renal ischemia/reperfusion. Thus, neutrophil PAD4 plays a pivotal role in renal ischemia/reperfusion-induced AKI.

Neutrophil Extracellular Traps as a Drug Target to Counteract Chronic and Acute Inflammation.

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.