Showing papers by "Konstantinos Ritis published in 2021"

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

Abstract: In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Patients with COVID-19: in the dark-NETs of neutrophils.

Abstract: SARS-CoV-2 infection poses a major threat to the lungs and multiple other organs, occasionally causing death. Until effective vaccines are developed to curb the pandemic, it is paramount to define the mechanisms and develop protective therapies to prevent organ dysfunction in patients with COVID-19. Individuals that develop severe manifestations have signs of dysregulated innate and adaptive immune responses. Emerging evidence implicates neutrophils and the disbalance between neutrophil extracellular trap (NET) formation and degradation plays a central role in the pathophysiology of inflammation, coagulopathy, organ damage, and immunothrombosis that characterize severe cases of COVID-19. Here, we discuss the evidence supporting a role for NETs in COVID-19 manifestations and present putative mechanisms, by which NETs promote tissue injury and immunothrombosis. We present therapeutic strategies, which have been successful in the treatment of immunο-inflammatory disorders and which target dysregulated NET formation or degradation, as potential approaches that may benefit patients with severe COVID-19.

Angiotensin II triggers neutrophil extracellular traps release linking thromboinflammation with essential hypertension.

Abstract: Innate immunity and chronic inflammation are involved in atherosclerosis and atherothrombosis leading to target organ damage in essential hypertension (EH). However, the role of neutrophils in EH is still elusive. We investigated the association between angiotensin II (Ang II) and neutrophil extracellular traps (NETs) in pathogenesis of EH. Plasma samples, kidney biopsies and surgical specimens of abdominal aortic aneurysms (AAA) from EH patients were used. Cell-based assays, NETs/human aortic endothelial cells co-cultures and in situ studies were performed. Increased plasma levels of NETs and tissue factor (TF) activity were detected in untreated, newly-diagnosed, EH patients. Stimulation of control neutrophils with plasma from untreated EH patients generated TF-enriched NETs promoting endothelial collagen production. Ang II induced NETosis in vitro via a reactive oxygen species (ROS)/peptidylarginine deiminase type 4 and autophagy-dependent pathway. Circulating NETs and thrombin generation levels were reduced significantly in EH patients starting treatment with Ang II receptor blockers, whereas their plasma was unable to trigger procoagulant NETs. Moreover, TF-bearing NETotic neutrophils/remnants were accumulated in sites of interstitial renal fibrosis and in the subendothelial layer of AAA. These data reveal the important pathogenic role of Ang II/ROS/NETs/TF axis in EH, linking thromboinflammation with endothelial dysfunction and fibrosis.

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

Abstract: 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 towards 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-1β. IL-17A-enriched AS NETs mediated the differentiation of MSCs towards bone-forming cells. The neutrophil expression of IL-17A was positively regulated by IL-1β. Blocking IL-1β 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 towards bone-forming cells. Moreover, IL-1β triggers the expression of IL-17A on NETs offering an additional therapeutic target in AS. This article is protected by copyright. All rights reserved.

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

Abstract: OBJECTIVES To explore the presence of neutrophil extracellular traps (NETs) in inflamed temporal artery biopsies (TABs) of patients with giant cell arteritis (GCA). METHODS Ten patients with GCA [5 with limited and 5 with associated generalized vascular involvement, as defined by 18F-fluorodeoxyglucose (FDG) positron-emission tomography with computed tomography (PET/CT)] and 8 with polymyalgia rheumatica (PMR) were studied. The presence, location, quantitation, and decoration of NETs with IL-6, IL-1β, 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-1β(+)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 centers will show whether the findings regarding neutrophils/NETs in the TAB are consistent and disclose their clinical impact.

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

Abstract: 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.

Neutrophil Extracellular Traps and Interleukin 17 in Ankylosing Spondylitis

Abstract: 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-1β 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-1β are expressed over those NETs and if NETs affect new bone formation.