Eirini Apostolidou

Neutrophil Extracellular Trap Formation Is Associated with IL-1β and Autophagy-Related Signaling in Gout

Background Gout is a prevalent inflammatory arthritis affecting 1–2% of adults characterized by activation of innate immune cells by monosodium urate (MSU) crystals resulting in the secretion of interleukin-1β (IL-1β). Since neutrophils play a major role in gout we sought to determine whether their activation may involve the formation of proinflammatory neutrophil extracellular traps (NETs) in relation to autophagy and IL-1β. Methodology/Principal Findings Synovial fluid neutrophils from six patients with gout crisis and peripheral blood neutrophils from six patients with acute gout and six control subjects were isolated. MSU crystals, as well as synovial fluid or serum obtained from patients with acute gout, were used for the treatment of control neutrophils. NET formation was assessed using immunofluorescence microscopy. MSU crystals or synovial fluid or serum from patients induced NET formation in control neutrophils. Importantly, NET production was observed in neutrophils isolated from synovial fluid or peripheral blood from patients with acute gout. NETs contained the alarmin high mobility group box 1 (HMGB1) supporting their pro-inflammatory potential. Inhibition of phosphatidylinositol 3-kinase signaling or phagolysosomal fusion prevented NET formation, implicating autophagy in this process. NET formation was driven at least in part by IL-1β as demonstrated by experiments involving IL-1β and its inhibitor anakinra. Conclusions/Significance These findings document for the first time that activation of neutrophils in gout is associated with the formation of proinflammatory NETs and links this process to both autophagy and IL-1β. Modulation of the autophagic machinery may represent an additional therapeutic study in crystalline arthritides.

Autophagy mediates the delivery of thrombogenic tissue factor to neutrophil extracellular traps in human sepsis.

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.

Tissue factor expression in neutrophil extracellular traps and neutrophil derived microparticles in antineutrophil cytoplasmic antibody associated vasculitis may promote thromboinflammation and the thrombophilic state associated with the disease

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.

Expression of functional tissue factor by neutrophil extracellular traps in culprit artery of acute myocardial infarction

Neutrophils are involved in the pathophysiology of infracted coronary arteries in STEMI via NET structures. Platelets, activated by thrombin, are required for NET formation, while the integrity of NET scaffold contributes to the functionality of NET-bound TF. The blockage of NET formation or local neutralization of NET-mediated TF signalling constitutes candidate therapeutic targets.

Neutrophil extracellular traps promote differentiation and function of fibroblasts.

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. Copyright

Neutrophil extracellular traps regulate IL-1β-mediated inflammation in familial Mediterranean fever

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.

Regulated in development and DNA damage responses 1 (REDD1) links stress with IL-1β–mediated familial Mediterranean fever attack through autophagy-driven neutrophil extracellular traps

Background Familial Mediterranean fever (FMF) is an IL‐1&bgr;–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‐1&bgr;–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 Stills 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‐1&bgr; 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‐1&bgr; 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‐1&bgr;, and might constitute an important piece in the IL‐1&bgr;–mediated inflammation puzzle. Graphical abstract Figure. No Caption available.

THU0394 Long-Term Therapy with Canakinumab in Two Patients with Refractory Chronic Autoinflammatory Arthritis

Background Canakinumab, a human anti-IL-1β monoclonal antibody, has been licensed for the treatment of cryopyrin-associated periodic syndrome (CAPS), while few reports have shown benefit in patients with refractory familial Mediterranean fever (FMF). However, clinical experience regarding long-term administration of canakinumab in rare autoinflammatory syndromes, such as FMF, has not been described to date1,2. Objectives To report the long-term efficacy and safety of canakinumab in two adults with chronic arthritis associated with mutations in the MEFV gene, refractory to conventional treatments. Methods Two patients with refractory chronic autoinflammatory arthritis were treated with canakinumab for a total of 42 and 24 months, respectively. The first patient (P1) was a 25 year-old female, homozygous for the MEFV M694V mutation, suffering from typical FMF with additional severe destructive arthritis of both hips and the left knee. Her arthritis had been resistant to colchicine, prednisolone, methotrexate and etanercept, while anakinra elicited severe injection site reactions2. The second patient (P2) was a 26 year-old female, with clinical characteristics shared between CAPS and FMF, homozygous for the R202Q mutation in MEFV. She suffered from chronic inflammatory polyarthritis resistant to colchicine and glucocorticoids and, although she had initially responded to anakinra, the drug was discontinued due to hypersensitivity reactions. Background colchicine was maintained in both cases. Clinical assessments, VAS pain/morning stiffness of the affected joints, VAS global assessment and acute phase reactants (CRP, ESR) were recorded every 4 weeks. MRI of the affected joints were performed at baseline and after treatment initiation. Results In P1 canakinumab was administered at a dose of 150 mg at weeks 0, 8, 14, 20 and every 4 weeks thereafter. The dose interval was shortened aiming to keep the patient symptom-free for the whole inter-dose interval. In P2 canakinumab was administered at a dose of 150 mg/8 weeks. Both patients soon experienced a significant improvement of the articular symptoms and remained in clinical remission during the rest of treatment. Neither patient experienced any systemic inflammatory attack during the treatment period. Further, canakinumab produced a normalization of acute phase reactants in P2 and moderate reductions in P1. In P1 follow-up MRI demonstrated focal areas of bone marrow edema, thickened synovium and chronic subchondral degenerative changes, which, however, remained stable over the 3-year period. In P2 complete radiological remission was evident on MRI. In both patients, canakinumab treatment allowed colchicine reduction to a minimal dosage. Overall, canakinumab was well tolerated and no adverse events were noted. Conclusions Our report suggests that canakinumab is effective and safe in patients with difficult to treat chronic autoinflammatory arthritis. These observations encourage the conduct of prospective clinical trials of canakinumab in FMF. References Soriano A, et al. Clin Rev Allergy Immunol. 2013;45:117. Mitroulis I, et al. Ann Rheum Dis. 2011;70:1347. Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.2249

CT findings of pulmonary involvement in antiphospholipid syndrome.

Aims: Antiphospholipid syndrome (APS) is a systemic autoimmune disorder characterized by vascular thrombosis. Pulmonary changes regarding interstitium and

THU0051 Tissue Factor Expression in Neutrophil Extracellular Traps in Patients with Active ANCA Associated Vasculitis

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