A fluorophore-conjugated reagent enabling rapid detection, isolation and live tracking of senescent cells.

Cellular senescence is a stress-response mechanism implicated in various physiological processes, diseases, and aging. Current detection approaches have partially addressed the issue of senescent cell identification in clinical specimens. Effective methodologies enabling precise isolation or live tracking of senescent cells are still lacking. In-depth analysis of truly senescent cells is, therefore, an extremely challenging task. We report (1) the synthesis and validation of a fluorophore-conjugated, Sudan Black-B analog (GLF16), suitable for in vivo and in vitro analysis of senescence by fluorescence microscopy and flow cytometry and (2) the development and application of a GLF16-carrying micelle vector facilitating GLF16 uptake by living senescent cells in vivo and in vitro. The compound and the applied methodology render isolation of senescent cells an easy, rapid, and precise process. Straightforward nanocarrier-mediated GLF16 delivery in live senescent cells comprises a unique tool for characterization of senescence at an unprecedented depth.

Senescent cells in giant cell arteritis display an inflammatory phenotype participating in tissue injury via IL-6-dependent pathways.

OBJECTIVES: Age is the strongest risk factor of giant cell arteritis (GCA), implying a possible pathogenetic role of cellular senescence. To address this question, we applied an established senescence specific multimarker algorithm in temporal artery biopsies (TABs) of GCA patients. METHODS: 75(+) TABs from GCA patients, 22(-) TABs from polymyalgia rheumatica (PMR) patients and 10(-) TABs from non-GCA/non-PMR patients were retrospectively retrieved and analysed. Synovial tissue specimens from patients with inflammatory arthritis and aorta tissue were used as disease control samples. Senescent cells and their histological origin were identified with specific cellular markers; IL-6 and MMP-9 were investigated as components of the senescent associated secretory phenotype by triple costaining. GCA or PMR artery culture supernatants were applied to fibroblasts, HUVECs and monocytes with or without IL-6R blocking agent to explore the induction of IL-6-associated cellular senescence. RESULTS: Senescent cells were present in GCA arteries at higher proportion compared with PMR (9.50% vs 2.66%, respectively, p<0.0001) and were mainly originated from fibroblasts, macrophages and endothelial cells. IL-6 was expressed by senescent fibroblasts, and macrophages while MMP-9 by senescent fibroblasts only. IL-6(+) senescent cells were associated with the extension of vascular inflammation (transmural inflammation vs adventitia limited disease: 10.02% vs 4.37%, respectively, p<0.0001). GCA but not PMR artery culture supernatant could induce IL-6-associated senescence that was partially inhibited by IL-6R blockade. CONCLUSIONS: Senescent cells with inflammatory phenotype are present in GCA arteries and are associated with the tissue inflammatory bulk, suggesting a potential implication in disease pathogenesis.

Down-regulation of KLF2 in lung fibroblasts is linked with COVID-19 immunofibrosis and restored by combined inhibition of NETs, JAK-1/2 and IL-6 signaling.

Kruppel-like factor 2 (KLF2) has been linked with fibrosis and neutrophil-associated thromboinflammation; however, its role in COVID-19 remains elusive. We investigated the effect of disease microenvironment on the fibrotic potential of human lung fibroblasts (LFs) and its association with KLF2 expression. LFs stimulated with plasma from severe COVID-19 patients down-regulated KLF2 expression at mRNA/protein and functional level acquiring a pre-fibrotic phenotype, as indicated by increased CCN2/collagen levels. Pre-incubation with the COMBI-treatment-agents (DNase I and JAKs/IL-6 inhibitors baricitinib/tocilizumab) restored KLF2 levels of LFs to normal abolishing their fibrotic activity. LFs stimulated with plasma from COMBI-treated patients at day-7 expressed lower CCN2 and higher KLF2 levels, compared to plasma prior-to-treatment, an effect not observed in standard-of-care treatment. In line with this, COMBI-treated patients had better outcome than standard-of-care group. These data link fibroblast KLF2 with NETosis and JAK/IL-6 signaling, suggesting the potential of combined therapeutic strategies in immunofibrotic diseases, such as COVID-19.

Limitations of Serological Diagnosis of Typical Cat Scratch Disease and Recommendations for the Diagnostic Procedure.

Introduction: Cat scratch disease (CSD) is the most common cause of bacterial infectious lymphadenopathy, especially in children, but its diagnosis still remains challenging. Serological assays are widely applied due to their simplicity and the non-invasive sampling. However, these techniques present several limitations, including not well-defined antigen preparation, assay conditions and cutoff titers, severe cross-reactions with other species and organisms, and the notably ranging seroprevalence in the normal population. The objective of this study is to review the literature in order to determine the best diagnostic procedure for the diagnosis of CSD. Methods: Databases including PubMed, Medline, Google Scholar, and Google were searched to determine the best diagnostic procedure for the diagnosis of CSD. A total of 437 papers were identified and screened, and after exclusion of papers that did not fulfill the including criteria, 63 papers were used. Results: It was revealed that sensitivities of serological assays varied from 10% to 100%. Indeed, more than half of the studies reported a sensitivity lower than 70%, while 71% of them had a sensitivity lower than 80%. Moreover, specificities of serological assays ranged from 15% to 100%, with 25 assays reporting a specificity lower than 90%. Conclusion: It is considered that molecular assays should be the gold standard technique for CSD confirmation, and physicians are reinforced to proceed to lymph node biopsy in suspicious CSD cases.

Neutrophil extracellular traps in giant cell arteritis biopsies: presentation, localization and co-expression with inflammatory cytokines.

OBJECTIVES: To explore the presence of neutrophil extracellular traps (NETs) in inflamed temporal artery biopsies (TABs) of patients with GCA. METHODS: Ten patients with GCA [five with limited and five with associated generalized vascular involvement, as defined by 18F-fluorodeoxyglucose PET with CT (PET/CT)] and eight with PMR were studied. The presence, location, quantitation and decoration of NETs with IL-6, IL-1ß and IL-17A were assessed in TABs at the time of disease diagnosis by tissue immunofluorescence and confocal microscopy. Paired serum levels of IL-6 and IL-17A were also evaluated in all patients. RESULTS: All temporal artery biopsies from GCA, but not PMR, patients had NETs located mainly in the adventitia, adjacent to the vasa vasorum. NETs decorated with IL-6 were present in 8/10 TABs of GCA patients, of whom 5 were PET/CT(+) and 3 PET/CT(-) patients. IL-17A(+) NETs were observed in all GCA patients. IL-1ß(+) NETs were not detected in any GCA patient. No relation was found between serum IL-6 and IL-17A levels and NETs containing IL-6 and/or IL-17A. CONCLUSIONS: NETs bearing pro-inflammatory cytokines are present in inflamed GCA-TABs. Future studies with a larger number of patients from different centres will show whether the findings regarding neutrophils/NETs in the TAB are consistent and disclose their clinical impact.

Fundamentally different roles of neuronal TNF receptors in CNS pathology: TNFR1 and IKKß promote microglial responses and tissue injury in demyelination while TNFR2 protects against excitotoxicity in mice.

BACKGROUND: During inflammatory demyelination, TNF receptor 1 (TNFR1) mediates detrimental proinflammatory effects of soluble TNF (solTNF), whereas TNFR2 mediates beneficial effects of transmembrane TNF (tmTNF) through oligodendroglia, microglia, and possibly other cell types. This model supports the use of selective inhibitors of solTNF/TNFR1 as anti-inflammatory drugs for central nervous system (CNS) diseases. A potential obstacle is the neuroprotective effect of solTNF pretreatment described in cultured neurons, but the relevance in vivo is unknown. METHODS: To address this question, we generated mice with neuron-specific depletion of TNFR1, TNFR2, or inhibitor of NF-κB kinase subunit ß (IKKß), a main downstream mediator of TNFR signaling, and applied experimental models of inflammatory demyelination and acute and preconditioning glutamate excitotoxicity. We also investigated the molecular and cellular requirements of solTNF neuroprotection by generating astrocyte-neuron co-cultures with different combinations of wild-type (WT) and TNF and TNFR knockout cells and measuring N-methyl-D-aspartate (NMDA) excitotoxicity in vitro. RESULTS: Neither neuronal TNFR1 nor TNFR2 protected mice during inflammatory demyelination. In fact, both neuronal TNFR1 and neuronal IKKß promoted microglial responses and tissue injury, and TNFR1 was further required for oligodendrocyte loss and axonal damage in cuprizone-induced demyelination. In contrast, neuronal TNFR2 increased preconditioning protection in a kainic acid (KA) excitotoxicity model in mice and limited hippocampal neuron death. The protective effects of neuronal TNFR2 observed in vivo were further investigated in vitro. As previously described, pretreatment of astrocyte-neuron co-cultures with solTNF (and therefore TNFR1) protected them against NMDA excitotoxicity. However, protection was dependent on astrocyte, not neuronal TNFR1, on astrocyte tmTNF-neuronal TNFR2 interactions, and was reproduced by a TNFR2 agonist. CONCLUSIONS: These results demonstrate that neuronal TNF receptors perform fundamentally different roles in CNS pathology in vivo, with neuronal TNFR1 and IKKß promoting microglial inflammation and neurotoxicity in demyelination, and neuronal TNFR2 mediating neuroprotection in excitotoxicity. They also reveal that previously described neuroprotective effects of solTNF against glutamate excitotoxicity in vitro are indirect and mediated via astrocyte tmTNF-neuron TNFR2 interactions. These results consolidate the concept that selective inhibition of solTNF/TNFR1 with maintenance of TNFR2 function would have combined anti-inflammatory and neuroprotective properties required for safe treatment of CNS diseases.

Exploiting the Role of Hypoxia-Inducible Factor 1 and Pseudohypoxia in the Myelodysplastic Syndrome Pathophysiology.

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of clonal hematopoietic stem (HSCs) and/or progenitor cells disorders. The established dependence of MDS progenitors on the hypoxic bone marrow (BM) microenvironment turned scientific interests to the transcription factor hypoxia-inducible factor 1 (HIF-1). HIF-1 facilitates quiescence maintenance and regulates differentiation by manipulating HSCs metabolism, being thus an appealing research target. Therefore, we examine the aberrant HIF-1 stabilization in BMs from MDS patients and controls (CTRLs). Using a nitroimidazole-indocyanine conjugate, we show that HIF-1 aberrant expression and transcription activity is oxygen independent, establishing the phenomenon of pseudohypoxia in MDS BM. Next, we examine mitochondrial quality and quantity along with levels of autophagy in the differentiating myeloid lineage isolated from fresh BM MDS and CTRL aspirates given that both phenomena are HIF-1 dependent. We show that the mitophagy of abnormal mitochondria and autophagic death are prominently featured in the MDS myeloid lineage, their severity increasing with intra-BM blast counts. Finally, we use in vitro cultured CD34+ HSCs isolated from fresh human BM aspirates to manipulate HIF-1 expression and examine its potential as a therapeutic target. We find that despite being cultured under 21% FiO2, HIF-1 remained aberrantly stable in all MDS cultures. Inhibition of the HIF-1α subunit had a variable beneficial effect in all <5%-intra-BM blasts-MDS, while it had no effect in CTRLs or in ≥5%-intra-BM blasts-MDS that uniformly died within 3 days of culture. We conclude that HIF-1 and pseudohypoxia are prominently featured in MDS pathobiology, and their manipulation has some potential in the therapeutics of benign MDS.

REDD1/Autophagy Pathway Is Associated with Neutrophil-Driven IL-1ß 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-1ß and thrombogenic tissue factor. The inflammatory environment of UC is able to induce neutrophil activation, IL-1ß 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-1ß 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.

REDD1/autophagy pathway promotes thromboinflammation and fibrosis in human systemic lupus erythematosus (SLE) through NETs decorated with tissue factor (TF) and interleukin-17A (IL-17A).

OBJECTIVES: The 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. METHODS: NET 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. RESULTS: Neutrophils 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. CONCLUSIONS: Our 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.

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 © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

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

BACKGROUND: Familial Mediterranean fever (FMF) is an IL-1ß-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-1ß-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-1ß 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-1ß 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-1ß, and might constitute an important piece in the IL-1ß-mediated inflammation puzzle.

Neutrophil extracellular traps exacerbate Th1-mediated autoimmune responses in rheumatoid arthritis by promoting DC maturation.

Aberrant formation of neutrophil extracellular traps (NETs) is a key feature in rheumatoid arthritis (RA) and plays a pivotal role in disease pathogenesis. However, the mechanism through which NETs shape the autoimmune response in RA remains elusive. In this study, we demonstrate that inhibition of peptidylarginine deiminases activity in collagen-induced arthritis (CIA) mouse model significantly reduces NET formation, attenuates clinical disease activity, and prevents joint destruction. Importantly, peptidylarginine deiminase 4 blocking markedly reduces the frequency of collagen-specific IFN-γ-producing T helper 1 (Th1) cells in the draining lymph nodes of immunized mice. Exposure of dendritic cells (DCs) to CIA-derived NETs induces DC maturation characterized by significant upregulation of costimulatory molecules, as well as elevated secretion of IL-6. Moreover, CIA-NET-treated DCs promote the induction of antigen-specific Th1 cells in vitro. Finally, NETs from RA patients show an increased potential to induce the maturation of DCs from healthy individuals, corroborating the findings obtained in CIA mouse model. Collectively, our findings delineate an important role of NETs in the induction and expansion of Th1 pathogenic cells in CIA through maturation of DCs and reveal a novel role of NETs in shaping the RA-autoimmune response that could be exploited therapeutically.

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

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.

Neutrophil extracellular traps regulate IL-1ß-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-1ß inhibitors. We investigated the role of neutrophil extracellular traps (NET) in the pathogenesis of FMF, and their involvement in IL-1ß 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-1ß 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-1ß production in PMNs and IL-1ß activity in NETs, respectively. CONCLUSIONS: We suggest two additive events for triggering the FMF attack; the production of IL-1ß 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.

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

AIMS: Neutrophil extracellular traps (NETs) are chromatin filaments released by activated polymorphonuclear neutrophils (PMNs) and decorated with granule proteins with various properties. Several lines of evidence implicate NETs in thrombosis. The functional significance and the in vivo relevance of NETs during atherothrombosis in humans have not been addressed until now. METHODS AND RESULTS: Selective sampling of thrombotic material and surrounding blood from the infarct-related coronary artery (IRA) and the non-IRA was performed during primary percutaneous revascularization in 18 patients with ST-segment elevation acute myocardial infarction (STEMI). Thrombi isolated from IRA contained PMNs and NETs decorated with tissue factor (TF). Although TF was expressed intracellularly in circulating PMNs of STEMI patients, active TF was specifically exposed by NETs obtained from the site of plaque rupture. Treatment of NET structures with DNase I abolished TF functionality measurement. In vitro treatment of control PMNs with plasma obtained from IRA and non-IRA was further shown to induce intracellular up-regulation of TF but not NET formation. A second step consisting of the interaction between PMNs and thrombin-activated platelets was required for NET generation and subsequent TF exposure. CONCLUSION: The interaction of thrombin-activated platelets with PMNs at the site of plaque rupture during acute STEMI results in local NET formation and delivery of active TF. The notion that NETs represent a mechanism by which PMNs release thrombogenic signals during atherothrombosis may offer novel 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.

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.

Current Insights into Tissue Injury of Giant Cell Arteritis: From Acute Inflammatory Responses towards Inappropriate Tissue Remodeling.

Giant cell arteritis (GCA) is an autoimmune disease affecting large vessels in patients over 50 years old. It is an exemplary model of a classic inflammatory disorder with IL-6 playing the leading role. The main comorbidities that may appear acutely or chronically are vascular occlusion leading to blindness and thoracic aorta aneurysm formation, respectively. The tissue inflammatory bulk is expressed as acute or chronic delayed-type hypersensitivity reactions, the latter being apparent by giant cell formation. The activated monocytes/macrophages are associated with pronounced Th1 and Th17 responses. B-cells and neutrophils also participate in the inflammatory lesion. However, the exact order of appearance and mechanistic interactions between cells are hindered by the lack of cellular and molecular information from early disease stages and accurate experimental models. Recently, senescent cells and neutrophil extracellular traps have been described in tissue lesions. These structures can remain in tissues for a prolonged period, potentially favoring inflammatory responses and tissue remodeling. In this review, current advances in GCA pathogenesis are discussed in different inflammatory phases. Through the description of these-often overlapping-phases, cells, molecules, and small lipid mediators with pathogenetic potential are described.

L-Dopa decarboxylase modulates autophagy in hepatocytes and is implicated in dengue virus-caused inhibition of autophagy completion.

The enzyme L-Dopa Decarboxylase (DDC) synthesizes the catecholamine dopamine and the indolamine serotonin. Apart from its role in the brain as a neurotransmitter biosynthetic enzyme, DDC has been detected also in the liver and other peripheral organs, where it is implicated in cell proliferation, apoptosis, and host-virus interactions. Dengue virus (DENV) suppresses DDC expression at the later stages of infection, during which DENV also inhibits autophagosome-lysosome fusion. As dopamine affects autophagy in neuronal cells, we investigated the possible association of DDC with autophagy in human hepatocytes and examined whether DDC mediates the relationship between DENV infection and autophagy. We performed DDC silencing/overexpression and evaluated autophagic markers upon induction of autophagy, or suppression of autophagosome-lysosome fusion. Our results showed that DDC favored the autophagic process, at least in part, through its biosynthetic function, while knockdown of DDC or inhibition of DDC enzymatic activity prevented autophagy completion. In turn, autophagy induction upregulated DDC, while autophagy reduction by chemical or genetic (ATG14L knockout) ways caused the opposite effect. This study also implicated DDC with the cellular energetic status, as DDC silencing reduced the oxidative phosphorylation activity of the cell. We also report that upon DDC silencing, the repressive effect of DENV on the completion of autophagy was enhanced, and the inhibition of autolysosome formation did not exert an additive effect on viral proliferation. These data unravel a novel role of DDC in the autophagic process and suggest that DENV downregulates DDC expression to inhibit the completion of autophagy, reinforcing the importance of this protein in viral infections.

One-step rapid tracking and isolation of senescent cells in cellular systems, tissues, or animal models via GLF16.

Identification and isolation of senescent cells is challenging, rendering their detailed analysis an unmet need. We describe a precise one-step protocol to fluorescently label senescent cells, for flow cytometry and fluorescence microscopy, implementing a fluorophore-conjugated Sudan Black-B analog, GLF16. Also, a micelle-based approach allows identification of senescent cells in vivo and in vitro, enabling live-cell sorting for downstream analyses and live in vivo tracking. Our protocols are applicable to cellular systems, tissues, or animal models where senescence is present. For complete details on the use and execution of this protocol, please refer to Magkouta et al.1.