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.

IL-17A expressed on neutrophil extracellular traps promotes mesenchymal stem cell differentiation toward bone-forming cells in ankylosing spondylitis.

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 toward 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-1ß. IL-17A-enriched AS NETs mediated the differentiation of MSCs toward bone-forming cells. The neutrophil expression of IL-17A was positively regulated by IL-1ß. Blocking IL-1ß 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 toward bone-forming cells. Moreover, IL-1ß triggers the expression of IL-17A on NETs offering an additional therapeutic target in AS.

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.

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.

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 and Interleukin 17 in Ankylosing Spondylitis.

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-1ß 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-1ß are expressed over those NETs and if NETs affect new bone formation.

Complement and tissue factor-enriched neutrophil extracellular traps are key drivers in COVID-19 immunothrombosis.

Emerging data indicate that complement and neutrophils contribute to the maladaptive immune response that fuels hyperinflammation and thrombotic microangiopathy, thereby increasing coronavirus 2019 (COVID-19) mortality. Here, we investigated how complement interacts with the platelet/neutrophil extracellular traps (NETs)/thrombin axis, using COVID-19 specimens, cell-based inhibition studies, and NET/human aortic endothelial cell (HAEC) cocultures. Increased plasma levels of NETs, tissue factor (TF) activity, and sC5b-9 were detected in patients. Neutrophils of patients yielded high TF expression and released NETs carrying active TF. Treatment of control neutrophils with COVID-19 platelet-rich plasma generated TF-bearing NETs that induced thrombotic activity of HAECs. Thrombin or NETosis inhibition or C5aR1 blockade attenuated platelet-mediated NET-driven thrombogenicity. COVID-19 serum induced complement activation in vitro, consistent with high complement activity in clinical samples. Complement C3 inhibition with compstatin Cp40 disrupted TF expression in neutrophils. In conclusion, we provide a mechanistic basis for a pivotal role of complement and NETs in COVID-19 immunothrombosis. This study supports strategies against severe acute respiratory syndrome coronavirus 2 that exploit complement or NETosis inhibition.