Stella Arelaki

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

Gradient Infiltration of Neutrophil Extracellular Traps in Colon Cancer and Evidence for Their Involvement in Tumour Growth

Background The role of neutrophils in tumour biology is largely unresolved. Recently, independent studies indicated either neutrophil extracellular traps (NETs) or Tissue Factor (TF) involvement in cancer biology and associated thrombosis. However, their individual or combined role in colonic adenocarcinoma is still unexplored. Methods Colectomy tissue specimens and variable number of draining lymph nodes were obtained from ten patients with adenocarcinoma of the colon. NETs deposition and neutrophil presence as well as TF expression were examined by immunostaining. The effect of NETs on cancer cell growth was studied in in vitro co-cultures of Caco-2 cell line and acute myeloid leukemia primary cells. Proliferation and apoptosis/necrosis of cancer cells were analyzed by flow cytometry. Results TF-bearing NETs and neutrophil localization were prominent in tumour sections and the respective metastatic lymph nodes. Interestingly, neutrophil infiltration and NETs concentration were gradually reduced from the tumour mass to the distal margin. The in vitro-generated NETs impeded growth of cancer cell cultures by inducing apoptosis and/or inhibiting proliferation. Conclusions These data support further the role of neutrophils and NETs in cancer biology. We also suggest their involvement on cancer cell growth.

Interferon lambda1/IL-29 and inorganic polyphosphate are novel regulators of neutrophil-driven thromboinflammation

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

NETopathies? Unraveling the Dark Side of Old Diseases through Neutrophils

Neutrophil extracellular traps (NETs) were initially described as an antimicrobial mechanism of neutrophils. Over the last decade, several lines of evidence support the involvement of NETs in a plethora of pathological conditions. Clinical and experimental data indicate that NET release constitutes a shared mechanism, which is involved in a different degree in various manifestations of non-infectious diseases. Even though the backbone of NETs is similar, there are differences in their protein load in different diseases, which represent alterations in neutrophil protein expression in distinct disorder-specific microenvironments. The characterization of NET protein load in different NET-driven disorders could be of significant diagnostic and/or therapeutic value. Additionally, it will provide further evidence for the role of NETs in disease pathogenesis, and it will enable the characterization of disorders in which neutrophils and NET-dependent inflammation are of critical importance.

Mast cells co-expressing CD68 and inorganic polyphosphate are linked with colorectal cancer

Inflammation is a hallmark of colorectal cancer (CRC). Neutrophils are well-known mediators in tumor biology but their role in solid tumors, including CRC, was redefined by neutrophil extracellular traps (NETs). Given that it was recently demonstrated that platelet-derived polyP primes neutrophils to release NETs, we examined surgical specimens from CRC to investigate the presence of polyP, as a possible NET inducer. Biopsies with adenomas, hyperplastic polyps, inflammatory bowel disease and healthy colon tissues were used as controls. In all cases, the presence of polyP was apparent, with the main source of polyP being the mast cells. In all CRC and all adenomas with high-grade dysplasia, a substantial number of mast cells, more than 50%, co-expressed intracellularly polyP with CD68 surface antigen (CD68+), but this was not the case in the other examined disorders. PolyP-expressing mast cells were detected in close proximity with tumor cells and neutrophils, suggesting polyP expression by CD68+ mast cells among the stimuli which prime neutrophils to release NETs, in CRC. Moreover, the detection of CD68+ polyP-expressing mast cells could represent a potential prognostic marker in colorectal adenomas and/or carcinomas.

REDD1/Autophagy Pathway Is Associated with Neutrophil-Driven IL-1β Inflammatory Response in Active Ulcerative Colitis

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.

Differential effect of hypoxia and acidity on lung cancer cell and fibroblast metabolism

This study examined the metabolic response of lung cancer cells and normal lung fibroblasts to hypoxia and acidity. GLUT1 and HXKII mRNA/protein expression was up-regulated under hypoxia in the MRC5 fibroblasts and in the A549 and H1299 lung cancer cell lines, indicating intensified glucose absorption and glycolysis. Under hypoxia, the LDHA mRNA and LDH5 protein levels increased in the cancer cells but not in the fibroblasts. Acidity suppressed the above-mentioned hypoxia effect. PDH-kinase-1 (PDK1 mRNA and protein) and inactive phosphorylated-PDH protein levels were induced under hypoxia in the cancer cells, whereas these were reduced in the MRC5 lung fibroblasts. In human tissue sections, the prevalent expression patterns supported the contrasting metabolic behavior of cancer cells vs. tumor fibroblasts. The monocarboxylate/lactate transporter 1 (MCT1) was up-regulated in all the cell lines under hypoxic conditions, but it was suppressed under acidic conditions. The mitochondrial DNA (mtDNA) content per cell decreased significantly in the A549 cancer cell line under hypoxia, but it increased in the MRC5 fibroblasts. Taking into account these findings, we suggest that, under hypoxia, cancer cells intensify the anaerobic direction in glycolysis, while normal fibroblasts prefer to seek energy by intensifying the aerobic use of the available oxygen.

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