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Complement C3 activation contributes to COVID-19 pathology, and C3 targeting has emerged as a promising therapeutic strategy. We provide interim data from ITHACA, the first randomized trial evaluating a C3 inhibitor, AMY-101, in severe COVID-19 (PaO2/FiO2 ≤ 300 mmHg). Patients received AMY-101 ( n = 16) or placebo ( n = 15) in addition to standard of care. AMY-101 was safe and well tolerated. Compared to placebo (8 of 15, 53.3%), a higher, albeit nonsignificant, proportion of AMY-101–treated patients (13 of 16, 81.3%) were free of supplemental oxygen at day 14. Three nonresponders and two placebo-treated patients succumbed to disease-related complications. AMY-101 significantly reduced CRP and ferritin and restrained thrombin and NET generation. Complete and sustained C3 inhibition was observed in all responders. Residual C3 activity in the three nonresponders suggested the presence of a convertase-independent C3 activation pathway overriding the drug’s inhibitory activity. These findings support the design of larger trials exploring the potential of C3-based inhibition in COVID-19 or other complement-mediated diseases.

Ankylosing spondylitis (AS) is an inflammatory disease characterized by excessive bone formation. We investigated the presence of neutrophil extracellular traps (NETs) in AS and how they are involved in the osteogenic capacity of bone marrow mesenchymal stem cells (MSCs) through interleukin-17A (IL-17A). Peripheral neutrophils and sera were obtained from patients with active AS and healthy controls. NET formation and neutrophil/NET-associated proteins were studied using immunofluorescence, immunoblotting, qPCR and ELISA. In vitro co-culture systems of AS NET structures and MSCs isolated from controls were deployed to examine the role of NETs in the differentiation of MSCs towards osteogenic cells. Analysis was performed using specific staining and qPCR. Neutrophils from patients with AS were characterized by enhanced formation of NETs carrying bioactive IL-17A and IL-1β. IL-17A-enriched AS NETs mediated the differentiation of MSCs towards bone-forming cells. The neutrophil expression of IL-17A was positively regulated by IL-1β. Blocking IL-1β signaling on neutrophils with anakinra or dismantling NETs using DNase-I disrupted osteogenesis driven by IL-17A-bearing NETs. These findings propose a novel role of neutrophils in AS-related inflammation, linking IL-17A-decorated NETs with the differentiation of MSCs towards bone-forming cells. Moreover, IL-1β triggers the expression of IL-17A on NETs offering an additional therapeutic target in AS. This article is protected by copyright. All rights reserved.

COVID-19-related severe respiratory failure (SRF) leads to mechanical ventilation increasing the in-hospital mortality substantially. Abundancy of lung fibroblasts (LFs) in injured lung tissue has been associated with the progression of respiratory failure in COVID-19. Aiming to reduce mortality in patients with SRF (PaO2/FiO2

Several studies have indicated that a neutrophil extracellular trap (NET) formation, apart from its role in host defense, can contribute to or drive pathogenesis in a wide range of inflammatory and thrombotic disorders. Therefore, NETs may serve as a therapeutic target or/and a diagnostic tool. Here, we compare the most commonly used techniques for the assessment of NET formation. Furthermore, we review recent data from the literature on the application of basic laboratory tools for detecting NET release and discuss the challenges and the advantages of these strategies in NET evaluation. Taken together, we provide some important insights into the qualitative and quantitative molecular analysis of NETs in translational medicine today.

Η ίνωση είναι μια αρκετά κοινή παθολογική κατάσταση, η οποία προσβάλει διάφορα όργανα και ιστούς. Αποτελεί την ανατομική βάση πολλών ασθενειών και είναι υπεύθυνη για αυξημένη θνησιμότητα σε όλες τις σύγχρονες κοινωνίες. Το εξωγενές σύστημα πήξης εμπλέκεται σε καταστάσεις ίνωσης μέσω της παραγωγής θρομβίνης. Η σηματοδότηση της θρομβίνης μέσω του PAR-1 υποδοχέα διαδραματίζει καθοριστικό ρόλο στην παθογένεια των φλεγμονωδών νοσημάτων και του καρκίνου. Παράλληλα, η συνομιλία μεταξύ φλεγμονής και εξωγενούς συστήματος πήξης έχει περιγραφεί σε αρκετά μοντέλα νόσων. Σημαντικό ρόλο στην ίνωση διαδραματίζουν οι ενεργοποιημένοι ινοβλάστες (μυοϊνοβλάστες), ενώ η ενδοθηλίνη-1 (ΕΤ-1) επάγει το σχηματισμό και την ενεργοποίηση των κυττάρων. Έτσι, για τη μελέτη του μηχανισμού της ίνωσης, έχοντας ως άξονα τη φλεγμονή και το εξωγενές σύστημα πήξης, επιλέχθηκε το μοντέλο της βρογχοπνευμονικής δυσπλασίας (BPD) σε νεογνά και το μοντέλο του συστηματικού σκληροδέρματος (SSc). Το BPD είναι μία ινωτική διαταραχή απειλητική για τη ζωή του νεογνού, η οποία εξελίσσεται ταχύτατα. Τα ουδετερόφιλα, που ανιχνεύονται στο κυψελιδικό μικροπεριβάλλον των νεογνών με BPD φαίνεται να παίζουν σημαντικό ρόλο στις φλεγμονώδεις αποκρίσεις της νόσου. Με βάση τα προηγούμενα ευρήματα του Εργαστηρίου Μοριακής Αιματολογίας σε σχέση με το μοντέλο της βρογχοπνευμονικής δυσπλασίας και τη συμμετοχή του εξωγενούς συστήματος πήξης σε ζωικά μοντέλα πνευμονικής ίνωσης, διερευνήσαμε το ρόλο του εξωγενούς συστήματος πήξης στις ινωτικές εκδηλώσεις της βρογχοπνευμονικής δυσπλασίας των νεογνών. Οι μυοϊνοβλάστες αποτέλεσαν τον κυτταρικό πληθυσμό-στόχο, ενώ το υπερκείμενο του BALF από νεογνά με BPD αποτέλεσε το βασικό διεγέρτη όλων των πειραμάτων (case group). Ως control group χρησιμοποιήθηκε το υπερκείμενο του BALF από νεογνά με σύνδρομο αναπνευστικής δυσχέρειας (RDS). Μυοϊνοβλάστες εντέρου από υγιή δότη χρησιμοποιήθηκαν για τα πειράματα αυτού του μοντέλου, ενώ τα πιο κρίσιμα αποτελέσματα επιβεβαιώθηκαν σε επαναληπτικές δοκιμασίες με τη χρήση μυοϊνοβλαστών πνεύμονα. Διαπιστώθηκε ότι οι μυοϊνοβλάστες που επωάστηκαν με το υπερκείμενο του BALF από νεογνά με BPD εμφανίζουν αυξημένη ικανότητα μετανάστευσης, η οποία εξαρτάται από την αναφυλατοξίνη C5a και τη θρομβίνη. Επιπλέον, οι μυοϊνοβλάστες εκφράζουν ιστικό παράγοντα (TF), με τρόπο που ρυθμίζεται από την ενδοθηλίνη-1 και κατόπιν παράγουν αυξητικό παράγοντα συνδετικού ιστού (CCN2), μέσω PAR-1 σηματοδότησης. Στη συνέχεια, η μελέτη μας στράφηκε στη νόσο του συστηματικού σκληροδέρματος. Το SSc είναι ένα αυτοάνοσο νόσημα, το οποίο προσβάλλει το δέρμα καθώς και εσωτερικά όργανα. Η ενεργοποίηση των κυττάρων του ανοσοποιητικού, η αγγειακή βλάβη και η υπέρμετρη εναπόθεση ινώδους συνδετικού ιστού στα προσβαλλόμενα όργανα αποτελούν τα πιο βασικά χαρακτηριστικά της νόσου. Καθώς το εξωγενές σύστημα πήξης ενεργοποιείται στη συγκεκριμένη νόσο και η χορήγηση Βosentan περιορίζει σημαντικά την ίνωση του δέρματος των ασθενών με SSc, διερευνήσαμε τη συνομιλία μεταξύ του άξονα TF-θρομβίνης και της σηματοδότησης της ενδοθηλίνης-1 στην ινωτική δραστηριότητα που παρατηρείται στη νόσο. Ειδικότερα, μελετήσαμε τις ιδιότητες, τη συμπεριφορά και την εμπλοκή των μυοϊνοβλαστών στη νόσο του συστηματικού σκληροδέρματος. Πραγματοποιήσαμε μία σειρά πειραμάτων στα οποία συμμετείχαν μυοϊνοβλάστες από ασθενείς με σκληρόδερμα (SSc HCMFs-case group) και μυοϊνοβλάστες από υγιείς δότες (Control HCMFs-control group). Παρατηρήσαμε ότι οι μυοϊνοβλάστες των ασθενών με SSc εμφανίζουν αυξημένη παραγωγή CCN2, κολλαγόνου και αυξημένη μεταναστευτική ικανότητα. Η συμπεριφορά τους αυτή εξαρτάται άμεσα από τη θρομβίνη. Επιπλέον, παρατηρήσαμε ότι οι μυοϊνοβλάστες των ασθενών έχουν αυξημένη έκφραση TF. Διαπιστώσαμε ότι η ενεργοποίηση του εξωγενούς συστήματος πήξης αυξάνει την έκφραση του υποδοχέα Α της ενδοθηλίνης (ETA), η οποία εξαρτάται από τη θρομβίνη. Η υπερ-έκφραση του ETA συμβάλλει σε περαιτέρω αύξηση τόσο της έκφρασης του TF όσο και της ινωτικής δραστηριότητας των μυοϊνοβλαστών. Η χορήγηση του Bosentan, που δρα ανασταλτικά στη σηματοδότηση της ενδοθηλίνης-1, αναστέλλει σημαντικά την ινωτική δραστηριότητα των μυοϊνοβλαστών, η οποία είναι εξαρτώμενη από τον TF. Συνοψίζοντας, τόσο το εξωγενές σύστημα της πήξης όσο και η ET-1 συμμετέχουν καθοριστικά στις ινωτικές εκδηλώσεις του BPD και του SSc. Παράλληλα, υποδεικνύουν νέους θεραπευτικούς στόχους για την αντιμετώπιση των ινωτικών επιπλοκών των νόσων. Λαμβάνοντας τα αποτελέσματα σχετικά με το ρόλο της ίνωσης από τα προαναφερθέντα μοντέλα, το ενδιαφέρον μας στράφηκε στη διερεύνηση του ρόλου των εξωκυττάριων ουδετεροφιλικών δικτύων χρωματίνης (ΝΕΤs) στην ινωτική διαδικασία. Μελέτες του Εργαστηρίου Μοριακής Αιματολογίας και πολυάριθμα δεδομένα από άλλα ερευνητικά κέντρα υπέδειξαν τη συμμετοχή των NETs στη φλεγμονή, στην αυτοανοσία, στην αντιγονοπαρουσίαση και στον καρκίνο. Για να μελετηθεί η συμμετοχή των εξωκυττάριων ουδετεροφιλικών δικτύων χρωματίνης στην ινωτική διαδικασία χρησιμοποιήθηκαν παράγοντες που σχετίζονται με την ίνωση (όπως είναι το εκχύλισμα τσιγάρων καπνού (CSE), το πυρητικό μαγνήσιο και η μπλεομυκίνη), αλλά και μη ινωτικά μόρια (όπως είναι το PMA). To CSE, το πυρητικό μαγνήσιο και η μπλεομυκίνη σχετίζονται με διάφορα μοντέλα ίνωσης, στα οποία τα ουδετερόφιλα συμμετέχουν καθοριστικά. Ωστόσο ο μηχανισμός εκδήλωσής της ίνωσης παρουσία αυτών των παραγόντων αλλά και η συμμετοχή των ουδετεροφίλων στην εκδήλωσή της παραμένει ασαφής. Στα πλαίσια αυτής της μελέτης, διαπιστώθηκε ότι τα ινωτικά μόρια δεν επάγουν την ινωτική διαδικασία όταν παρέχονται άμεσα στου ινοβλάστες. Αντίθετα, τα ουδετερόφιλα από υγιείς δότες σχηματίζουν εξωκυττάρια ουδετεροφιλικά δίκτυα χρωματίνης ύστερα από επώαση με τους ινωτικούς παράγοντες. Διαπιστώθηκε μάλιστα ότι ο σχηματισμός των εξωκυττάριων ουδετεροφιλικών δικτύων χρωματίνης εξαρτάται από την αυτοφαγία και την κιτρουλινοποίηση των ιστονών. Επιπλέον, οι ινοβλάστες που επωάζονται με τις δομές των ουδετεροφίλων [οι οποίες σχηματίζονται είτε από τα παραπάνω ινωτικά μόρια είτε από μη-ινωτικούς διεγέρτες (ΡΜΑ)], ενεργοποιούνται, διαφοροποιούνται σε μυοϊνοβλάστες (ενεργοποιημένοι ινοβλάστες) και εκδηλώνουν ινωτική συμπεριφορά. Η συμμετοχή των εξωκυττάριων ουδετεροφιλικών δικτύων χρωματίνης στη διαφοροποίηση/ενεργοποίηση των ινοβλαστών διαπιστώθηκε με ειδική αναστολή της χρωματίνης και των ιστονών, με τη χρήση DNase I και ηπαρίνης αντίστοιχα. Επιπλέον, η IL-17, η οποία αποτελεί εναρκτήριο μόριο της φλεγμονής/ίνωσης, εντοπίστηκε πάνω στα ΝΕΤs που απελευθερώνονται παρουσία αυτών των ινωτικών ή μη παραγόντων. Συγκεκριμένα, διαπιστώσαμε ότι IL-17 διαδραματίζει σημαντικό ρόλο στην ινωτική συμπεριφορά των ήδη διαφοροποιημένων ινοβλαστών (μυοϊνοβλάστες). Tα παραπάνω αποτελέσματα επιβεβαιώθηκαν σε ανθρώπινο πνευμονικό ιστό με (εκτεταμένη) ίνωση, όπου εντοπίστηκαν εξωκυττάρια δίκτυα χρωματίνης σε εστίες με ίνωση και σε εγγύς θέσεις με ενεργοποιημένους ινοβλάστες. Τα ευρήματα αυτά φανερώνουν τον καθοριστικό ρόλο των ουδετεροφίλων στο μηχανισμό της ίνωσης και παρέχουν αρκετά σημεία για θεραπευτικές παρεμβάσεις.

ObjectivesThe release of neutrophil extracellular traps (NETs) represents a novel neutrophil effector function in systemic lupus erythematosus (SLE) pathogenesis. However, the molecular mechanism underlying NET release and how NETs mediate end-organ injury in SLE remain elusive.MethodsNET formation and NET-related proteins were assessed in the peripheral blood and biopsies from discoid lupus and proliferative nephritis, using immunofluorescence, immunoblotting, quantitative PCR and ELISA. Autophagy was assessed by immunofluorescence and immunoblotting. The functional effects of NETs in vitro were assessed in a primary fibroblast culture.ResultsNeutrophils from patients with active SLE exhibited increased basal autophagy levels leading to enhanced NET release, which was inhibited in vitro by hydroxychloroquine. NETosis in SLE neutrophils correlated with increased expression of the stress-response protein REDD1. Endothelin-1 (ET-1) and hypoxia-inducible factor-1α (HIF-1α) were key mediators of REDD1-driven NETs as demonstrated by their inhibition with bosentan and L-ascorbic acid, respectively. SLE NETs were decorated with tissue factor (TF) and interleukin-17A (IL-17A), which promoted thrombin generation and the fibrotic potential of cultured skin fibroblasts. Notably, TF-bearing and IL-17A-bearing NETs were abundant in discoid skin lesions and in the glomerular and tubulointerstitial compartment of proliferative nephritis biopsy specimens.ConclusionsOur data suggest the involvement of REDD1/autophagy/NET axis in end-organ injury and fibrosis in SLE, a likely candidate for repositioning of existing drugs for SLE therapy. Autophagy-mediated release of TF-bearing and IL-17A-bearing NETs provides a link between thromboinflammation and fibrosis in SLE and may account for the salutary effects of hydroxychloroquine.

Innate immunity and chronic inflammation are involved in atherosclerosis and atherothrombosis leading to target organ damage in essential hypertension (EH). However, the role of neutrophils in EH is still elusive. We investigated the association between angiotensin II (Ang II) and neutrophil extracellular traps (NETs) in pathogenesis of EH. Plasma samples, kidney biopsies and surgical specimens of abdominal aortic aneurysms (AAA) from EH patients were used. Cell-based assays, NETs/human aortic endothelial cells co-cultures and in situ studies were performed. Increased plasma levels of NETs and tissue factor (TF) activity were detected in untreated, newly-diagnosed, EH patients. Stimulation of control neutrophils with plasma from untreated EH patients generated TF-enriched NETs promoting endothelial collagen production. Ang II induced NETosis in vitro via a reactive oxygen species (ROS)/peptidylarginine deiminase type 4 and autophagy-dependent pathway. Circulating NETs and thrombin generation levels were reduced significantly in EH patients starting treatment with Ang II receptor blockers, whereas their plasma was unable to trigger procoagulant NETs. Moreover, TF-bearing NETotic neutrophils/remnants were accumulated in sites of interstitial renal fibrosis and in the subendothelial layer of AAA. These data reveal the important pathogenic role of Ang II/ROS/NETs/TF axis in EH, linking thromboinflammation with endothelial dysfunction and fibrosis.

Emerging data indicate that complement and neutrophils are involved in the maladaptive host immune response that fuels hyper-inflammation and thrombotic microangiopathy increasing the mortality rate in coronavirus disease 2019 (COVID-19). Here, we investigated the interaction between complement and the platelet/neutrophil extracellular traps (NETs)/thrombin axis, using COVID-19 clinical samples, cell-based inhibition studies and NETs/human aortic endothelial cell (HAEC) co-cultures. Increased plasma levels of NETs, TF activity and sC5b-9 were detected in patients. Neutrophils yielded high tissue factor (TF) expression and released NETs carrying functionally active TF. Confirming our ex vivo findings, treatment of control neutrophils with COVID-19 platelet-rich plasma generated TF-bearing NETs that induced thrombotic activity of HAEC. Thrombin or NETosis inhibition or C5aR1 blockade attenuated platelet-mediated NET-driven thrombogenicity. Serum isolated from COVID-19 patients induces complement activation in vitro, which is consistent with high complement activity in clinical samples. Complement inhibition at the level of C3 with compstatin Cp40 disrupted TF expression in neutrophils. In conclusion, we provide a mechanistic basis that reveals the pivotal role of complement and NETs in COVID-19 immmunothrombosis. This study supports emerging strategies against SARS-CoV-2 infection that exploit complement therapeutics or NETosis inhibition.

Neutrophils through the release of neutrophil extracellular traps (NETs) containing active tissue factor (TF) are key components of thrombo-inflammation. Platelets-neutrophils interplay in ST elevation myocardial infarction (STEMI) promotes NET formation via inorganic polyphosphates (polyP) released by thrombin-activated platelets. NETs, however, are also induced by biomaterials in a platelet-independent manner. Considering the possible pleiotropic effects of Ticagrelor beyond platelet inhibition and the clinical need for novel antithrombotic strategies targeting inflammation, we investigated the effects of Ticagrelor on polyP and stent-induced NETs in STEMI. Neutrophils from healthy individuals and patients receiving Ticagrelor were stimulated with polyP or drug-eluting stents (DES) to produce NETs. To induce TF expression, neutrophils were further incubated with plasma obtained from the infarct-related artery (IRA) of STEMI patients. The effects of Ticagrelor on NETs and TF loading were assessed using fluorescence microscopy, flow cytometry, myeloperoxidase(MPO)/DNA complex ELISA, and a Western blot. Ticagrelor interrupts platelet&ndash, neutrophil interaction by attenuating NETs induced by polyP. However, Ticagrelor does not affect polyP secretion from thrombin-activated platelets. Similarly, the intracellular production of TF in neutrophils triggered by IRA plasma is not hindered by Ticagrelor. Furthermore, DES induce NETs and synchronous stimulation with IRA plasma leads to the formation of thrombogenic TF-bearing NETs. Ticagrelor inhibits stent-induced NET release. These findings suggest a novel immune-modulatory effect of Ticagrelor when it attenuates the formation of thrombogenic NETs.

Growing clinical evidence has implicated complement as a pivotal driver of COVID-19 immunopathology. Deregulated complement activation may fuel cytokine-driven hyper-inflammation, thrombotic microangiopathy and NET-driven immunothrombosis, thereby leading to multi-organ failure. Complement therapeutics have gained traction as candidate drugs for countering the detrimental consequences of SARS-CoV-2 infection. Whether blockade of terminal complement effectors (C5, C5a, or C5aR1) may elicit similar outcomes to upstream intervention at the level of C3 remains debated. Here we compare the efficacy of the C5-targeting monoclonal antibody eculizumab with that of the compstatin-based C3-targeted drug candidate AMY-101 in small independent cohorts of severe COVID-19 patients. Our exploratory study indicates that therapeutic complement inhibition abrogates COVID-19 hyper-inflammation. Both C3 and C5 inhibitors elicit a robust anti-inflammatory response, reflected by a steep decline in C-reactive protein and IL-6 levels, marked lung function improvement, and resolution of SARS-CoV-2-associated acute respiratory distress syndrome (ARDS). C3 inhibition afforded broader therapeutic control in COVID-19 patients by attenuating both C3a and sC5b-9 generation and preventing FB consumption. This broader inhibitory profile was associated with a more robust decline of neutrophil counts, attenuated neutrophil extracellular trap (NET) release, faster serum LDH decline, and more prominent lymphocyte recovery. These early clinical results offer important insights into the differential mechanistic basis and underlying biology of C3 and C5 inhibition in COVID-19 and point to a broader pathogenic involvement of C3-mediated pathways in thromboinflammation. They also support the evaluation of these complement-targeting agents as COVID-19 therapeutics in large prospective trials., Highlights • Deregulated complement activation underpins the pathophysiology of severe COVID-19 • Complement inhibition abrogates COVID-19 hyper-inflammation leading to resolution of SARS-CoV-2-associated ARDS • Divergent profiles of C3 vs C5 inhibition point to a broader impact of C3 inhibition on NET-driven thromboinflammation

The co-existence of an autoinflammatory syndrome with a demyelinating disorder is a very rare occurrence raising the question whether there is a pathophysiological connection between them. We describe the case of a man with symptoms of cryopyrin-associated periodic syndrome (CAPS) since infancy who later developed multiple sclerosis (MS). As CAPS was genetically confirmed, the inhibition of interleukin-1 (IL-1) with anakinra led to a swift resolution of the CAPS symptoms and also, in combination with teriflunomide, to a clinical and imaging improvement of MS. In vitro studies showed that, upon a CAPS flare, the patient's peripheral neutrophils released neutrophil extracellular traps (NETs) decorated with IL-1β, while NET release was markedly decreased following anakinra-induced remission of CAPS. Taking into account the growing evidence on the involvement of IL-1β in experimental models of MS, this rare patient case suggests that the role of neutrophils/NETs and IL-1β in MS should be further studied.

Neutrophils and neutrophil-released meshwork structures termed neutrophil extracellular traps (NETs) are major mediators of thromboinflammation and emerging targets for therapy, yet the mechanisms and pathways that control the role of neutrophils in thromboinflammation remain poorly understood. Here, we explored the role of IFN-λ1/IL-29, a major antiviral cytokine recently shown to suppress the neutrophil migratory capacity, in prothrombotic and proNETotic functions of neutrophils. In an ex vivo human experimental setting of acute ST-segment elevation myocardial infarction (STEMI), we show that IFN-λ1/IL-29 hinders NET release and diminishes the amount of cytoplasmic TF in neutrophils. Since platelet-neutrophil interaction plays a major role in NET-induced thromboinflammation, we further studied how IFN-λ1/IL-29 may interrupt this interaction. In this context, we identified inorganic polyphosphate (polyP) as a platelet-derived NET inducer in STEMI. In arterial STEMI thrombi, polyP was present in platelets and in close proximity to NET remnants. PolyP release from activated platelets was dependent on thrombin present in infarcted artery plasma, resulting in NET formation by promoting mTOR inhibition and autophagy induction. The effect of polyP on mTOR inhibition was counteracted by IFN-λ1/IL-29 treatment, leading to inhibition of NET formation. Consistently, we show in an in vivo model of FeCl3 -induced arterial thrombosis that IFN-λ2/IL-28A exerts strong antithrombotic potential. Taken together, these findings reveal a novel function of IFN-λ1/IL-29 in the suppression of thromboinflammation. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Transplant-associated thrombotic microangiopathy (TA-TMA) is a severe and life-threatening complication of hematopoietic cell transplantation (HCT) that often coincides with graft-versus-host-disease (GVHD). Although endothelial damage seems to be the common denominator for both disorders, the role of complement system, neutrophils, and coagulation has not been clarified. In an effort to distinguish the pathogenesis of TA-TMA from GVHD, we evaluated markers of complement activation, neutrophil extracellular trap (NET) release, endothelial damage, and activation of coagulation cascade in the circulation of patients with these two disorders, as well as control HCT recipients without TA-TMA or GVHD. We observed that the terminal complement product C5b-9 levels, the levels of markers of NET formation, and thrombin–antithrombin complex levels were significantly increased in the TA-TMA group compared with patients without complications, whereas there was no significant difference between the GVHD and the control group. On the other hand, the levels of circulating thrombomodulin, an endothelial damage marker, were significantly increased in both TA-TMA and GVHD patients. These findings propose a role for the interplay between complement system, neutrophil activation through NET release, and activation of the coagulation cascade in TA-TMA.

Ankylosing spondylitis (AS) is a chronic inflammatory disease traditionally regarded as mediated by T lymphocytes. Recent progress has identified that cells of innate immunity are also important for the processes of inflammation and new bone formation, a hallmark of AS. Moreover, interleukin-17 (IL-17) is a cytokine implicated in both processes. Neutrophils are increasingly recognized as mediators of autoinflammatory and autoimmune diseases through several mechanisms, one being the release of neutrophil extracellular traps (NETs). NETs are equipped with an array of bioactive molecules, such as IL-1β or IL-17. It appears that the molecules expressed over NETs vary across different disorders, reflecting diverse pathophysiologic mechanisms. As few studies have investigated the role of neutrophils in AS, the purpose of this research protocol is to study whether neutrophils from AS patients are more likely to form NETs, whether IL-17 and IL-1β are expressed over those NETs and if NETs affect new bone formation.

Infiltration of neutrophils into colonic mucosa has been associated with the severity of ulcerative colitis (UC). We investigated the effect of disease microenvironment on the release of neutrophil extracellular traps (NETs) as well as the involved mechanisms in NETosis and whether certain NET proteins are correlated with disease phenotype. Peripheral blood neutrophils, sera, and colonic tissue were collected from treatment-naive and mesalazine-treated patients with active UC, treatment-naive patients with active Crohn’s disease, patients suffering from infectious colitis, or healthy individuals (controls). Analysis of colonic biopsy specimens and peripheral blood neutrophils for the presence of NET-related markers using immunofluorescence confocal microscopy, ELISA, immunoblotting, flow cytometry, and quantitative PCR were performed. In vitro cell and tissue culture systems were further deployed. The local inflammatory response in colon in UC, but not Crohn’s disease, is characterized by the presence of NETs carrying bioactive IL-1β and thrombogenic tissue factor. The inflammatory environment of UC is able to induce neutrophil activation, IL-1β expression, and NET release, as shown both ex vivo and in vitro. REDD1 expression, as a mediator linking inflammation, autophagy, and NET release, was also specifically associated with the inflammatory response of UC. We show that neutrophil expression of REDD1 in colon tissue and the presence of IL-1β in neutrophils/NETs provide candidate biomarkers for the differential diagnosis of inflammatory colitis and possible targets for the treatment of UC, suggesting that UC shares common features with autoinflammatory disorders.

Neutrophil activation by inflammatory stimuli and the release of extracellular chromatin structures (neutrophil extracellular traps - NETs) have been implicated in inflammatory disorders. Herein, we demonstrate that NETs released by neutrophils treated either with fibrosis-related agents, such as cigarette smoke, magnesium silicate, bleomycin, or with generic NET inducers, such as phorbol 12-myristate 13-acetate, induced activation of lung fibroblasts (LFs) and differentiation into myofibroblast (MF) phenotype. Interestingly, the aforementioned agents or IL-17 (a primary initiator of inflammation/fibrosis) had no direct effect on LF activation and differentiation. MFs treated with NETs demonstrated increased connective tissue growth factor expression, collagen production, and proliferation/migration. These fibrotic effects were significantly decreased after degradation of NETs with DNase1, heparin or myeloperoxidase inhibitor, indicating the key role of NET-derived components in LF differentiation and function. Furthermore, IL-17 was expressed in NETs and promoted the fibrotic activity of differentiated LFs but not their differentiation, suggesting that priming by DNA and histones is essential for IL-17-driven fibrosis. Additionally, autophagy was identified as the orchestrator of NET formation, as shown by inhibition studies using bafilomycin A1 or wortmannin. The above findings were further supported by the detection of NETs in close proximity to alpha-smooth muscle actin (α-SMA)-expressing fibroblasts in biopsies from patients with fibrotic interstitial lung disease or from skin scar tissue. Together, these data suggest that both autophagy and NETs are involved not only in inflammation but also in the ensuing fibrosis and thus may represent potential therapeutic targets in human fibrotic diseases.

Neutrophils and neutrophil-released meshwork structures termed neutrophil extracellular traps (NETs) are major mediators of thromboinflammation and emerging targets for therapy, yet the mechanisms and pathways that control the role of neutrophils in thromboinflammation remain poorly understood. Here, we explored the role of IFN-λ1/IL-29, a major antiviral cytokine recently shown to suppress the neutrophil migratory capacity, in prothrombotic and proNETotic functions of neutrophils. In an ex vivo human experimental setting of acute ST-segment elevation myocardial infarction (STEMI), we show that IFN-λ1/IL-29 hinders NET release and diminishes the amount of cytoplasmic TF in neutrophils. Since platelet-neutrophil interaction plays a major role in NET-induced thromboinflammation, we further studied how IFN-λ1/IL-29 may interrupt this interaction. In this context, we identified inorganic polyphosphate (polyP) as a platelet-derived NET inducer in STEMI. In arterial STEMI thrombi, polyP was present in platelets and in close proximity to NET remnants. PolyP release from activated platelets was dependent on thrombin present in infarcted artery plasma, resulting in NET formation by promoting mTOR inhibition and autophagy induction. The effect of polyP on mTOR inhibition was counteracted by IFN-λ1/IL-29 treatment, leading to inhibition of NET formation. Consistently, we show in an in vivo model of FeCl

Background Familial Mediterranean fever (FMF) is an IL-1β–dependent autoinflammatory disease caused by mutations of Mediterranean fever (MEFV) encoding pyrin and characterized by inflammatory attacks induced by physical or psychological stress. Objective We investigated the underlying mechanism that links stress-induced inflammatory attacks with neutrophil activation and release of IL-1β–bearing neutrophil extracellular traps (NETs) in patients with FMF. Methods RNA sequencing was performed in peripheral neutrophils from 3 patients with FMF isolated both during attacks and remission, 8 patients in remission, and 8 healthy subjects. NET formation and proteins were analyzed by using confocal immunofluorescence microscopy, immunoblotting, myeloperoxidase-DNA complex ELISA, and flow cytometry. Samples from patients with Still's disease and bacterial infections were used also. Results The stress-related protein regulated in development and DNA damage responses 1 (REDD1) is significantly overexpressed during FMF attacks. Neutrophils from patients with FMF during remission are resistant to autophagy-mediated NET release, which can be overcome through REDD1 induction. Stress-related mediators (eg, epinephrine) decrease this threshold, leading to autophagy-driven NET release, whereas the synchronous inflammatory environment of FMF attack leads to intracellular production of IL-1β and its release through NETs. REDD1 in autolysosomes colocalizes with pyrin and nucleotide-binding domain, leucine-rich repeat/pyrin domain-containing 3. Mutated pyrin prohibits this colocalization, leading to higher IL-1β levels on NETs. Conclusions This study provides a link between stress and initiation of inflammatory attacks in patients with FMF. REDD1 emerges as a regulator of neutrophil function upstream to pyrin, is involved in NET release and regulation of IL-1β, and might constitute an important piece in the IL-1β–mediated inflammation puzzle.

Familial Mediterranean fever (FMF) is an autoinflammatory disease, characterized by MEFV gene mutations and self-limited recurrent episodes of fever and localized serositis. Complement system is a key regulator of the inflammatory process. The aim of this study was to investigate the genetic alterations and mRNA expression pattern of C5aR and C5L2 genes in neutrophils from attack-free FMF patients. No mutations were observed in the two receptors' genes, while the genetic alteration observed in the C5aR1 gene was identified as N279 K polymorphic variant. Furthermore, lower mRNA expression of C5L2 gene was observed in neutrophils from FMF patients compared to control subjects. The binding capacity of rhC5a and the ability to produce reactive oxygen species was similar in neutrophils from healthy subjects and FMF patients and independent of the presence of N279 K polymorphism or mRNA expression of C5L2.

Neonatal respiratory distress syndrome can progress to bronchopulmonary dysplasia (BPD), a serious pulmonary fibrotic disorder. Given the involvement of the extrinsic coagulation cascade in animal models of lung fibrosis, we examined its role in BPD. We observed a higher number of neutrophils expressing tissue factor (TF) in bronchoalveolar lavage fluid (BALF) from infants with BPD than from those with uncomplicated respiratory distress syndrome together with a parallel decrease in TF and connective tissue growth factor (CTGF) in BALF supernatants during the disease course. The involvement of coagulation in the fibrotic process associated with BPD was further evaluated by treating primary human colonic myofibroblasts with BALF supernatants from infants with BPD. These human colonic myofibroblasts demonstrated an enhanced C5a- and thrombin-dependent migration. Moreover, they expressed TF in an endothelin-1–dependent manner, with subsequent activation of the extrinsic coagulation cascade and CTGF production mediated by protease-activator receptor-1 signaling. These data provide a novel mechanism for the development of BPD and indicate that endothelin-1 signaling contributes to fibrosis by upregulating a TF/thrombin amplification loop responsible for CTGF production, and offer novel and specific therapeutic targets for pulmonary fibrotic disease.

Familial Mediterranean fever (FMF) inflammatory attacks are often triggered by metabolic or physical stress. mTOR signaling and autophagy modulate cellular responses to metabolic danger signals. In this study, we investigated the implication of mTOR inhibition and autophagy in FMF pathophysiology. mTOR inhibition induced MEFV gene expression in polymorphonuclear cells (PMNs) from healthy individuals, whereas it had no effect on PMNs from attack-free FMF patients. A significant reduction in pyrin levels in PMNs from FMF patients after mTOR inhibition was also observed. Pyrin levels in control PMNs remained unaffected. Moreover, the basal autophagic status in PMNs from FMF patients was reduced, as indicated by the lower LC3B-II/I ratio and ATG mRNA expression levels. However, mTOR inhibition had similar effects on the induction of autophagy in the two groups. The differential pyrin expression after metabolic stress induction and the impaired basal autophagy suggest a potential role in the triggering of FMF attacks.

The screening for JAK2 V617F mutation in patients with polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis offers crucial information for the final diagnosis of these disorders. Recently, several JAK2 exons 12 and 14 mutations have been detected in V617F-negative patients with idiopathic erythrocytosis. The need for a rapid and accurate assay for the mutation screening in both exons 12 and 14 prompted us for the application of a method for the analysis of the entire coding region between exons 11 and 15. We applied the non-isotopic RNase cleavage assay and the accuracy of the method was verified in a series of V617F-positive, V617F-negative patients and healthy individuals, with no false results. This method can be applied in any laboratory without the requirement of specific sophisticated equipment.

Objective Inflammatory attacks of familial Mediterranean fever (FMF) are characterised by circulation and influx of high number of polymorphonuclear neutrophils (PMN) in the affected sites and profound therapeutic effect of IL-1β inhibitors. We investigated the role of neutrophil extracellular traps (NET) in the pathogenesis of FMF, and their involvement in IL-1β production. Methods Blood samples were obtained from six FMF patients during remissions and from three patients during attacks. NET formation and NET components were studied by fluorescence techniques, immunobloting and MPO-DNA complex ELISA. Results PMNs from patients released NETs decorated with IL-1β during disease attacks. On the other hand, PMNs from patients during remission were resistant to inflammatory stimuli that induce NET release in PMNs from control subjects. Lower basal autophagy levels were identified in PMNs during remission, while induction of autophagy facilitated NET release, suggesting that autophagy is involved in the regulation of NET release. During the resolution of attacks, inhibition of NET formation by negative feedback mechanism was also observed. The anti-inflammatory agents, colchicine and DNAse I, inhibited IL-1β production in PMNs and IL-1β activity in NETs, respectively. Conclusions We suggest two additive events for triggering the FMF attack; the production of IL-1β by PMNs and its release through NETs. At the same time NETs, homeostatically, downregulate further NETosis, facilitating the resolution of attack. Compensatorly, lower basal autophagy of PMNs may protect from crises by attenuating the release of pro-inflammatory NETs.

Neutrophil activation by inflammatory stimuli and the release of extracellular chromatin structures (neutrophil extracellular traps - NETs) have been implicated in inflammatory disorders. Herein, we demonstrate that NETs released by neutrophils treated either with fibrosis-related agents, such as cigarette smoke, magnesium silicate, bleomycin, or with generic NET inducers, such as phorbol 12-myristate 13-acetate, induced activation of lung fibroblasts (LFs) and differentiation into myofibroblast (MF) phenotype. Interestingly, the aforementioned agents or IL-17 (a primary initiator of inflammation/fibrosis) had no direct effect on LF activation and differentiation. MFs treated with NETs demonstrated increased connective tissue growth factor expression, collagen production, and proliferation/migration. These fibrotic effects were significantly decreased after degradation of NETs with DNase1, heparin or myeloperoxidase inhibitor, indicating the key role of NET-derived components in LF differentiation and function. Furthermore, IL-17 was expressed in NETs and promoted the fibrotic activity of differentiated LFs but not their differentiation, suggesting that priming by DNA and histones is essential for IL-17-driven fibrosis. Additionally, autophagy was identified as the orchestrator of NET formation, as shown by inhibition studies using bafilomycin A1 or wortmannin. The above findings were further supported by the detection of NETs in close proximity to alpha-smooth muscle actin (α-SMA)-expressing fibroblasts in biopsies from patients with fibrotic interstitial lung disease or from skin scar tissue. Together, these data suggest that both autophagy and NETs are involved not only in inflammation but also in the ensuing fibrosis and thus may represent potential therapeutic targets in human fibrotic diseases.

Objectives Antineutrophil cytoplasmic antibody (ANCA) associated vasculitis (AAV) is characterised by neutrophil activation. An elevated prevalence of venous thromboembolic events has been reported in AAV. Because of the critical role of neutrophils in inflammation associated thrombosis, we asked whether neutrophil tissue factor (TF) may be implicated in the thrombotic diathesis in AAV. Methods Neutrophils from four patients and sera from 17 patients with ANCA associated vasculitis with active disease and remission were studied. TF expression was assessed by immunoblotting and confocal microscopy. Circulating DNA levels were evaluated. TF expressing microparticles (MPs) were measured by flow cytometry and thrombin–antithrombin complex levels by ELISA. Results Peripheral blood neutrophils from four patients with active disease expressed elevated TF levels and released TF expressing neutrophil extracellular traps (NETs) and MPs. TF positive NETs were released by neutrophils isolated from the bronchoalveolar lavage and were detected in nasal and renal biopsy specimens. Elevated levels of circulating DNA and TF expressing neutrophil derived MPs were further observed in sera from patients with active disease. Induction of remission attenuated the aforementioned effects. Control neutrophils treated with sera from patients with active disease released TF bearing NETs and MPs which were abolished after IgG depletion. Treatment of control neutrophils with isolated IgG from sera from patients with active disease also resulted in the release of TF bearing NETs. TF implication in MP dependent thrombin generation was demonstrated by antibody neutralisation studies. Conclusions Expression of TF in NETs and neutrophil derived MPs proposes a novel mechanism for the induction of thrombosis and inflammation in active AAV.

Background: Sepsis is associated with systemic inflammatory responses and induction of coagulation system. Neutrophil extracellular traps (NETs) constitute an antimicrobial mechanism, recently implicated in thrombosis via platelet entrapment and aggregation. Methodology/Principal Findings: In this study, we demonstrate for the first time the localization of thrombogenic tissue factor (TF) in NETs released by neutrophils derived from patients with gram-negative sepsis and normal neutrophils treated with either serum from septic patients or inflammatory mediators involved in the pathogenesis of sepsis. Localization of TF in acidified autophagosomes was observed during this process, as indicated by positive LC3B and LysoTracker staining. Moreover, phosphatidylinositol 3-kinase inhibition with 3-MA or inhibition of endosomal acidification with bafilomycin A1 hindered the release of TF-bearing NETs. TF present in NETs induced thrombin generation in culture supernatants, which further resulted in protease activated receptor-1 signaling. Conclusions/Significance: This study demonstrates the involvement of autophagic machinery in the extracellular delivery of TF in NETs and the subsequent activation of coagulation cascade, providing evidence for the implication of this process in coagulopathy and inflammatory response in sepsis. © 2012 Kambas et al.

Objective. The extrinsic coagulation cascade is involved in the fibrotic process, via thrombin-dependent induction of CCN2 (connective tissue growth factor) expression. Given the previously reported activation of the coagulation system in systemic sclerosis (SSc), we undertook the present study to investigate the involvement of cross-talk between the tissue factor (TF)– thrombin axis and endothelin 1 (ET-1) signaling in the fibrotic activity of SSc. Methods. Human colonic myofibroblasts (HCMFs) from 6 patients with SSc and gastrointestinal symptoms and from 6 control subjects were isolated and cultured under various conditions. Messenger RNA and protein levels of TF, CCN2, and endothelin receptor A (ETA) were investigated. Collagen production and migratory activity of HCMFs were further assessed. Results. HCMFs from SSc patients demonstrated increased basal CCN2 production, collagen deposition, and migration rate, in a thrombin-dependent manner. Increased TF expression was also observed in SSc HCMFs. Subsequent activation of the extrinsic coagulation system resulted in thrombin-dependent enhancement of ETA expression. ETA overexpression led to further increases in both TF expression and fibrotic activity in HCMFs. Moreover, inhibition of ET-1 signaling by bosentan abolished the TF-mediated fibrotic capacity of HCMFs. Conclusion. Tissue factor–thrombin signaling is involved in the increased fibrotic activity of HCMFs from patients with SSc. Moreover, the up-regulation of ET A expression by thrombin and the effect of ET-1 in the induction of TF expression indicate an amplification loop for enhanced collagen deposition. Therapeutic interventions targeting the extrinsic coagulation system or ET-1 signaling may provide clinical benefit by breaking this vicious circle.

The induction of the autophagy machinery, a process for the catabolism of cytosolic proteins and organelles, constitutes a crucial mechanism in innate immunity. However, the involvement of autophagy in human neutrophils and the possible inducers of this process have not been completely elucidated. In this study, the induction of autophagy was examined in human neutrophils treated with various activators and detected by the formation of acidified autophagosomes through monodansylcadaverine staining and via LC-3B conversion screened by immunoblotting and immunofluorescence confocal microscopy. In addition, the expression of the ATG genes was assessed by real-time RT-PCR. We provide evidence that autophagy is implicated in human neutrophils in both a phagocytosis-independent (rapamycin, TLR agonists, PMA) and phagocytosis (Escherichia coli)-dependent initiation manner. ROS activation is a positive mechanism for autophagy induction in the case of PMA, TLR activation and phagocytosis. Furthermore, LC3B gene expression was uniformly upregulated, indicating a transcriptional level of regulation for the autophagic machinery. This study provides a stepping stone toward further investigation of autophagy in neutrophil-driven inflammatory disorders.

Background ANCA-associated vasculitis (AAV) is a group of disorders characterized by neutrophil activation [1], an exuberant inflammatory response and hypercoagulability associated with a significant increase in the prevalence of venous thromboembolism [2]. Neutrophils may contribute to thromboinflammation through the release of neutrophil extracellular traps (NETs) and the expression of tissue factor (TF) [3]. Objectives To study the role of neutrophils in thromboinflammation in AAV. Methods Neutrophils from three patients and sera from 16 patients with ANCA-associated vasculitis with active disease and remission were collected. TF expression was assessed by immunoblotting in cell lysates and confocal microscopy in NETs. Serum DNA levels were also evaluated after staining with propidium iodide and fluorescence measurement with fluorometer [4]. Results Peripheral blood neutrophils from patients with active disease released NETs expressing TF visualized by confocal microscopy after staining with TF, elastase and DAPI. The activation of autophagy and the inclusion of TF in autophagosomes were further demonstrated with confocal microscopy, using the autophagy markers LC3, LysoTracker and p62-SQSTM1. Neutrophils isolated from the bronchoalveolar lavage from two patients also released TF-expressing NETs. Similar structures were also detected in nasal biopsy specimens from two patients, as indicated by the co-staining with elastase, TF and DAPI. Elevated levels of circulating DNA were further observed in sera from patients with active disease, indicating the in vivo release of NETs. TF expression, assessed by immunoblotting and release of TF expressing NETs were attenuated after successful induction of remission. Treatment of control neutrophils with sera from patients with active disease resulted in the formation of TF expressing NETs. This effect was abolished after IgG depletion. Conclusions This study demonstrates, for the first time, the expression of TF, the principal in vivo inducer of coagulation, in NETs during active AAV. TF-induced thrombin generation may activate protease activated receptor (PAR)-signaling and promote thrombosis and inflammation in active AAV. References Lane SE, et al. Primary systemic vasculitis: clinical features and mortality. QJM 2005;98:97–111. Allenbach Y, et al. High frequency of venous thromboembolic events in Churg-Strauss syndrome, Wegener’s granulomatosis and microscopic polyangiitis but not polyarteritis nodosa: a systematic retrospective study on 1130 patients. Ann Rheum Dis 2009;68:564-7. Kambas K, et al. The emerging role of neutrophils in thrombosis-the journey of TF through NETs. Front Immunol. 2012;3:385. Kambas K, et al. Autophagy mediates the delivery of thrombogenic tissue factor to neutrophil extracellular traps in human sepsis. PLoS One 2012;7:e45427. Disclosure of Interest None Declared