Theocharis Konstantinidis

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.

Immunomodulatory Role of Clarithromycin in Acinetobacter baumannii Infection via Formation of Neutrophil Extracellular Traps

ABSTRACT Macrolide antibiotics have been shown to act as immunomodulatory molecules in various immune cells. However, their effect on neutrophils has not been extensively investigated. In this study, we investigated the role of macrolide antibiotics in the generation of neutrophil extracellular traps (NETs). By assessing ex vivo and in vivo NET formation, we demonstrated that clarithromycin is able to induce NET generation both in vitro and in vivo. Clarithromycin utilizes autophagy in order to form NETs, and these NETs are decorated with antimicrobial peptide LL-37. Clarithromycin-induced NETs are able to inhibit Acinetobacter baumannii growth and biofilm formation in an LL-37-dependent manner. Additionally, LL-37 antimicrobial function depends on NET scaffold integrity. Collectively, these data expand the knowledge on the immunomodulatory role of macrolide antibiotics via the generation of LL-37-bearing NETs, which demonstrate LL-37-dependent antimicrobial activity and biofilm inhibition against A. baumannii.

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.

Autoantibodies in patients with asthma: is there a link between asthma and autoimmunity?

The objective of this study was to measure autoantibodies and the mean individual immune reactivity (IR) in children with asthma. The study group consisted of ten children with asthma and the control group of ten age-matched healthy subjects. In all patients IR and 24 autoantibodies (aAb) type G were measured by ELISA (Immunculus, Russia). The IR in asthmatics was 42% and was statistically significantly higher than this of control group 9% (p < 0.005). The level of aAbs (LuM, LuS, CoM and dsDNA) was also raised in the asthmatics. Anti-Adr aAbs was higher in one patient who required intense treatment for asthma with frequent use of corticosteroids. Anti-HMMP, insulin and IR aAbs were significantly lower in comparison to the control group (p < 0.05). This study confirms the high level of immunological activation in patients with asthma. The presence of autoantibodies implies that autoimmunity might have a role in the pathogenesis of asthma.

Gene promoter methylation and protein expression of BRMS1 in uterine cervix in relation to high-risk human papilloma virus infection and cancer.

Cervical cancer is strongly related to certain high-risk types of human papilloma virus infection. Breast cancer metastasis suppressor 1 (BRMS1) is a tumor suppressor gene, its expression being regulated by DNA promoter methylation in several types of cancers. This study aims to evaluate the methylation status of BRMS1 promoter in relation to high-risk types of human papilloma virus infection and the development of pre-cancerous lesions and describe the pattern of BRMS1 protein expression in normal, high-risk types of human papilloma virus–infected pre-cancerous and malignant cervical epithelium. We compared the methylation status of BRMS1 in cervical smears of 64 women with no infection by high-risk types of human papilloma virus to 70 women with proven high-risk types of human papilloma virus infection, using real-time methylation-specific polymerase chain reaction. The expression of BRMS1 protein was described by immunohistochemistry in biopsies from cervical cancer, pre-cancerous lesions, and normal cervices. Methylation of BRMS1 promoter was detected in 37.5% of women with no high-risk types of human papilloma virus infection and was less frequent in smears with high-risk types of human papilloma virus (11.4%) and in women with pathological histology (cervical intraepithelial neoplasia) (11.9%). Methylation was detected also in HeLa cervical cancer cells. Immunohistochemistry revealed nuclear BRMS1 protein staining in normal high-risk types of human papilloma virus–free cervix, in cervical intraepithelial neoplasias, and in malignant tissues, where staining was occasionally also cytoplasmic. In cancer, expression was stronger in the more differentiated cancer blasts. In conclusion, BRMS1 promoter methylation and aberrant protein expression seem to be related to high-risk types of human papilloma virus–induced carcinogenesis in uterine cervix and is worthy of further investigation.

PD46 - Serum level of S 100 proteins in patients with asthma

Aim Asthma is a chronic disease of the lower respiratory tract that is characterized by inflammation and bronchial obstruction. The pathophysiology of the disease is related to constriction of the bronchial smooth muscle, something that is influenced by calcium homeostasis. Important role in calcium homeostasis, play various binding proteins, which belongs to the family of proteins S100 (S100). The purpose of this study was to measure S100 protein in a group of children with bronchial asthma (BA) in comparison with age-sex matched control group.

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

Clarithromycin Enhances the Antibacterial Activity and Wound Healing Capacity in Type 2 Diabetes Mellitus by Increasing LL-37 Load on Neutrophil Extracellular Traps

Background: Type 2 diabetes mellitus (T2D) is characterized by susceptibility to bacterial infections and impaired wound healing. Neutrophil extracellular traps (NETs) and the cathelicidin antimicrobial peptide LL-37 have been implicated both in defense against bacterial infections and in wound healing process. Recently, it was shown that macrolide antibiotic clarithromycin induces the release of LL-37-bearing NETs. In T2D there has not been identified any link between NETs and LL-37 and the effect of clarithromycin in neutrophils/NETs is unknown yet. Methods: Peripheral blood neutrophils were obtained from treatment-naive hyperglycemic T2D patients (naive), normoglycemic T2D patients under antidiabetic treatment (well-controlled) and healthy donors (controls). NET release and NET proteins were studied. Co-culture systems of NET structures with E. coli NCTC 9001 and primary skin fibroblasts were deployed to examine the in vitro antibacterial and fibrotic NET properties, respectively. The effect of clarithromycin was also investigated. Analysis was performed using immunofluorescence confocal microscopy, myeloperoxidase-DNA complex and LL-37 ELISA, immunoblotting and qRT-PCR. Results: NETs were characterized by the presence of LL-37, however they lacked antibacterial activity, in both groups of T2D patients. Clarithromycin significantly increased the externalization of LL-37 on NETs generated from well-controlled T2D neutrophils, thus restoring NET antibacterial capacity and promoting the wound healing process via fibroblast activation and differentiation. Conclusion: This study suggests that clarithromycin may add further advantage to well-controlled T2D patients, by enhancing their antibacterial defense and improving wound healing capacity of fibroblasts, through upregulation of LL-37 on NET structures.