Macrophage IL-1β-positive microvesicles exhibit thrombo-inflammatory properties and are detectable in patients with active juvenile idiopathic arthritis

ObjectiveIL-1β is a leaderless cytokine with poorly known secretory mechanisms that is barely detectable in serum of patients, including those with an IL-1β-mediated disease such as systemic juvenile idiopathic arthritis (sJIA). Leukocyte microvesicles (MVs) may be a mechanism of IL-1β secretion. The first objective of our study was to characterize IL-1β-positive MVs obtained from macrophage cell culture supernatants and to investigate their biological functions in vitro and in vivo. The second objective was to detect circulating IL-1β-positive MVs in JIA patients.MethodsMVs were purified by serial centrifugations from PBMCs, or THP-1 differentiated into macrophages, then stimulated with LPS ± ATP. MV content was analyzed for the presence of IL-1β, NLRP3 inflammasome, caspase-1, P2X7 receptor, and tissue factor (TF) using ELISA, Western blot, or flow cytometry. MV biological properties were studied in vitro by measuring VCAM-1, ICAM-1, and E-selectin expression after HUVEC co-culture and factor-Xa generation test was realized. In vivo, MVs’ ability to recruit leukocytes in a murine model of peritonitis was evaluated. Plasmatic IL-1β-positive MVs were studied ex vivo in 10 active JIA patients using flow cytometry.ResultsTHP-1-derived macrophages stimulated with LPS and ATP released MVs, which contained NLRP3, caspase-1, and the 33-kDa precursor and 17-kDa mature forms of IL-1β and bioactive TF. IL-1β-positive MVs expressed P2X7 receptor and released soluble IL-1β in response to ATP stimulation in vitro. In mice, MVs induced a leukocyte peritoneal infiltrate, which was reduced by treatment with the IL-1 receptor antagonist. Finally, IL-1β-positive MVs were detectable in plasma from 10 active JIA patients.ConclusionMVs shed from activated macrophages contain IL-1β, NLRP3 inflammasome components, and TF, and constitute thrombo-inflammatory vectors that can be detected in the plasma from active JIA patients

Procoagulant and fibrinolytic activity of microvesicles, from their role in sepsis physiopathology to in vitro test development in hemostasis

Les microvésicules (MV) sont des vésicules extracellulaires submicroniques libérées à la suite du réarrangement des phospholipides membranaires en réponse à un stimulus extérieur ou à l'apoptose. Les MV sont des vecteurs bioactifs dont le rôle dans l'hémostase et la réponse inflammatoire est bien décrit à ce jour. La vision initiale attribuant aux MV une activité uniquement procoagulante a été récemment élargie par la démonstration d'une activité fibrinolytique. Cette thèse comporte deux volets, le premier s’intéresse à la mesure de l’activité procoagulante des MV (MV-FT), en optimisant les performances analytiques de la mesure de l’activité du facteur tissulaire des MV et un deuxième volet de cette thèse s’est intéressé à un nouveau mécanisme reliant la génération de plasmine des MV de granulocytes (Gran-MV) au pronostic des patients en choc septique (CS). Cette activité fibrinolytique implique le complexe entre l’urokinase (uPA) et son récepteur porté par les Gran-MV jouant un rôle de vecteur d'activité fibrinolytique. Cette activité thrombolytique s'intègre dans un rôle protecteur des MV capables de contrebalancer le risque de microthrombose systémique associé à la gravité dans le CS. Cette activité fibrinolytique identifie les MV non seulement comme biomarqueurs, mais également en tant qu’agent thérapeutiques potentielles. La maîtrise de ces tests fonctionnels d’activité de génération de plasmine des MV (MV-PGC) et d’activité MV-FT, utilisant les MV comme biomarqueurs dans l’évaluation du statut hémostatique du patient, est une étape essentielle pour accélérer le transfert de ces innovations diagnostiques et thérapeutiques au chevet du patient.Microvesicles (MV) are submicron extracellular vesicles released following the rearrangement of membrane phospholipids in response to an external stimulus or apoptosis. MV are bioactive vectors whose role in hemostasis and inflammatory response has been well described to date. The initial vision attributing to MV only procoagulant activity has recently been widened by the demonstration of fibrinolytic activity. This thesis has two parts, the first part focuses on the measurement of procoagulant activity of MV (MV-FT), by optimizing the analytical performance of measuring the activity of tissue factor on MV and a second part of this thesis was interested in a new mechanism linking the plasmin generation activity of MV from granulocytes (Gran-MV) to the prognosis of patients with septic shock (CS). This fibrinolytic activity involves urokinase (uPA) and its receptor carried by Gran-MV playing a role of vector of fibrinolytic activity. This thrombolytic activity is part of a protective role for MV capable of counterbalancing the risk of systemic microthrombosis associated with severity in CS. This fibrinolytic activity identifies MV not only as biomarkers, but also as potential therapeutic agents. These functional tests of MV plasmin generation activity (MV-PGC) and MV-FT activity, using MV as biomarkers in the evaluation of the patient's hemostatic status, is an essential step to accelerate the transfer of these diagnostic and therapeutic innovations to the bedside

Optimisation d’un test d’activité du facteur tissulaire sur les microvésicules

Le complexe formé entre le facteur tissulaire (FT) et le facteur VII est le principal activateur de la coagulation. Des niveaux élevés d'activité pro-coagulante dépendante du FT portée par les micro-vésicules (MV) sont détectables chez les patients présentant un risque accru de thrombose. Cependant, l’association entre l’activité du FT sur les MV (activité MV-FT) et la thrombose est limitée par de multiples mécanismes de thrombose liée aux cancers, un rôle plus complexe des MV dans la thrombose et enfin des limites technologiques dans le test mesurant l’activité MV FT. Plusieurs méthodes ont été décrites pour mesurer l'activité MV-FT, mais elles sont gênées par une sensibilité et une spécificité limitées. L'objectif est d'augmenter la sensibilité de l'analyse de l'activité de MV-FT en comparaison à un test préexistant (appelé test de Chapel Hill). L’amélioration du dosage de l'activité du FT a été réalisée en optimisant i/la vitesse et le temps de centrifugation, ii/l’utilisation d'un puissant anticorps anti-FT inhibiteur iii/l’utilisation de FVII et d'un substrat fluorogène pour augmenter la spécificité. La spécificité de mesure de l’activité MV-FT a été démontrée par l’absence d’activité sur une lignée de cellules qui expriment le FT en utilisant un anticorps monoclonal anti-FT appelé SBTF-1, qui présente un effet inhibiteur de FT supérieur à celui de l’anticorps monoclonal anti-FT humain, le plus utilisé dans la littérature, appelé HTF-1. Des expériences utilisant du sang provenant d'individus en bonne santé, stimulés ou non par le LPS, ou de plasma enrichi avec 3 niveaux différents de MV ont montré que le nouveau dosage était plus sensible et détecte de l'activité du FT dans les échantillons de plasma sans plaquettes (PFP) provenant d'individus sains. Cependant, le test a été limité par une variabilité inter test, principalement due à l'étape de centrifugation. Ainsi, nous avons amélioré la sensibilité de l'activité MV-FT sans perdre de spécificité. Ce nouveau test pourrait être utilisé pour évaluer les niveaux de MV-FT en tant que biomarqueur potentiel du risque thrombotique chez les patients

Microparticles and Fibrinolysis

International audienceMicroparticles (MPs) are submicronic vesicles which are formed by budding of the cellular membrane of virtually any cell type in response to cell activation or apoptosis. Both circulating MPs and MPs generated within tissues harbor molecules with a large repertoire of biological activities and transfer material to target cells. Depending on their cellular origin, the stimuli triggering their formation, or their localization, they may participate in the maintenance of organ or vascular homeostasis as well as inducing dysfunction. MPs have mostly been described as having procoagulant properties. However, the fact that some MP subsets are able to efficiently generate plasmin suggests that the role of MPs in hemostasis is more complex than initially thought. In this review, we summarize key findings showing that MPs provide a heterogeneous catalytic surface for plasmin generation, according to their cellular origin. We further address the specific features of the MP-dependent fibrinolytic system. Potential consequences of this MP-associated fibrinolytic activity in pathology are illustrated in cancer

A new hybrid immunocapture bioassay with improved reproducibility to measure tissue factor-dependent procoagulant activity of microvesicles from body fluids

International audienceBackground: The procoagulant activity of tissue factor-bearing microvesicles (MV-TF) has been associated with the risk of developing venous thrombosis in cancer patients. However, MV-TF assays are limited either by i) a lack of specificity, ii) a low sensitivity, or iii) a lack of repeatability when high-speed centrifugation (HS-C) is used to isolate MV. Therefore, our objective was to develop a new hybrid "capture-bioassay" with improved reproducibility combining MV immunocapture from biofluids and measurement of their TF activity.Materials and methods: Factor Xa generation and flow cytometry assays were used to evaluate IMS beads performance, and to select the most effective capture antibodies. The analytical performance between IMS-based and HS-C-based assays was evaluated with various models of plasma samples (from LPS-activated blood, spiked with tumoral MV, or with saliva MV) and different biofluids (buffer, plasma, saliva, and pleural fluid).Results: Combining both CD29 and CD59 antibodies on IMS beads was as efficient as HS-C to isolate plasmatic PS + MV. The IMS-based strategy gave significantly higher levels of MV-TF activity than HS-C in tumor MV spiked buffer, and both pleural fluids and saliva samples. Surprisingly, lower TF values were measured in plasma due to TFPI (TF pathway inhibitor) non-specifically adsorbed onto beads. This was overcome by adding a TFPI-blocking antibody. After optimization, the new IMS-based assay significantly improved reproducibility of MV-TF bioassay versus the HS-C-based assay without losing specificity and sensitivity. In addition, this approach could identify the cellular origin of MV-TF in various biological fluids.Conclusion: Compared to HS-C, the IMS-based measurement of MV-TF activity in body fluids improves reproducibility and makes the assay compatible with clinical practice. It can facilitate future automation

Microvesicles and Cancer Associated Thrombosis

Microvesicles (MVs) are small membrane enclosed structures released into the extracellular space by virtually all cell types. Their composition varies according to the cell origin and the stimulus which caused their formation. They harbor functional molecules and participate in intercellular communication. Endothelium, inflammatory cells, and cancer cells produce procoagulant MVs which contribute to cancer-associated thrombosis (CAT) in animal models. The tissue factor (TF) conveyed by these MVs was shown to play a key role in different animal models of experimental CAT. Alternatively, other molecular mechanisms involving polyphosphates or phosphatidylethanolamine could also be involved. In clinical practice, an association between an increase in the number of TF-positive or the procoagulant activity of these MVs and the occurrence of CAT has indeed been demonstrated in pancreatic-biliary cancers, suggesting that they could behave as a biomarker predictive for CAT. However, to date, this association was not confirmed in other types of cancer. Potential causes explaining this limited associated between MVs and CAT are (1) the diversity of mechanisms associating MVs and different types of cancer; (2) a more complex role of MVs in hemostasis integrating their anticoagulant and fibrinolytic activity; and (3) the lack of sensitivity, reproducibility, and standardization of current methodologies permitting measurement of MVs. Each of these hypotheses constitutes an interesting exploration path for a future reassessment of the clinical interest of the MVs in CAT

A new assay to evaluate microvesicle plasmin generation capacity: validation in disease with fibrinolysis imbalance

International audienceAmong extracellular vesicles, leukocyte-derived microvesicles (LMVs) have emerged as complex vesicular structures. Primarily identified as procoagulant entities, they were more recently ascribed to plasmin generation capacity (MV-PGC). The objectives of this work were (1) to develop a new hybrid bio-assay combining the specific isolation of LMVs and measurement of their PGC, and compare its performance to the original method based on centrifugation, (2) to validate MV-PGC in septic shock, combining increased levels of LMVs and fibrinolytic imbalance. Using plasma sample spiked with LMVs featuring different levels of PGC, we demonstrated that CD15-beads specifically extracted LMVs. The MV dependency of the test was demonstrated using electron microscopy, high speed centrifugation, nanofiltration and detergent-mediated solubilization and the MV-PGC specificity using plasmin-specific inhibitors, or antibodies blocking elastase or uPA. Thanks to a reaction booster (epsilon-ACA), we showed that the assay was more sensitive and reproducible than the original method. Moreover, it exhibited a good repeatability, inter-operator and inter-experiment reproducibility. The new immunomagnetic bio-assay was further validated in patients with septic shock. As a result, we showed that MV-PGC values were significantly lower in septic shock patients who died compared to patients who survived, both at inclusion and 24 h later (1.4 [0.8-3.0] vs 3.1 [1.7-18] A(405) x 10(-3)/min, p = 0.02; 1.4 [1-1.6] vs 5.2 [2.2-16] A(405) x 10(-3)/min, p = 0.004). Interestingly, combining both MV-PGC and PAI-1 in a ratio significantly improved the predictive value of PAI-1. This strategy, a hybrid capture bioassay to specifically measure LMV-PGC using for the first time, opens new perspectives for measuring subcellular fibrinolytic potential in clinical settings with fibrinolytic imbalance

A new strategy to count and sort neutrophil‐derived extracellular vesicles: Validation in infectious disorders

International audienceNewly recognized polymorphonuclear neutrophil (PMNs) functions include the ability to release subcellular mediators such as neutrophil-derived extracellular vesicles (NDEVs) involved in immune and thrombo-inflammatory responses. Elevation of their plasmatic level has been reported in a variety of infectious and cardiovascular disorders, but the clinical use of this potential biomarker is hampered by methodological issues. Although flow cytometry (FCM) is currently used to detect NDEVs in the plasma of patients, an extensive characterization of NDEVs has never been done. Moreover, their detection remains challenging because of their small size and low antigen density. Therefore, the objective of the present study was first to establish a surface antigenic signature of NDEVs detectable by FCM and therefore to improve their detection in biological fluids by developing a strategy allowing to overcome their low fluorescent signal and reduce the background noise. By testing a large panel of 54 antibody specificities already reported to be positive on PMNs, we identified a profile of 15 membrane protein markers, including 4 (CD157, CD24, CD65 and CD66c) never described on NDEVs. Among them, CD15, CD66b and CD66c were identified as the most sensitive and specific markers to detect NDEVs by FCM. Using this antigenic signature, we developed a new strategy combining the three best antibodies in a cocktail and reducing the background noise by size exclusion chromatography (SEC). This strategy allowed a significant improvement in NDEVs enumeration in plasma from sepsis patients and made it feasible to efficiently sort NDEVs from COVID-19 patients. Altogether, this work opens the door to a more valuable measurement of NDEVs as a potential biomarker in clinical practice. A similar strategy could also be applied to improve detection by FCM of other rare subpopulations of EVs generated by tissues with limited access, such as vascular endothelium, cancer cells or placenta

Increasing the sensitivity of the human microvesicle tissue factor activity assay

Introduction: The TF-FVIIa complex is the primary activator of coagulation. Elevated levels of microvesicle (MV) bearing tissue factor (TF)-dependent procoagulant activity are detectable in patients with an increased risk of thrombosis. Several methods have been described to measure MV TF activity but they are hampered by limited sensitivity and specificity. The aim of this work was to increase the sensitivity of the MV TF activity assay (called Chapel Hill assay).Material and methods: Improvements of the MV TF activity assay included i/ speed and time of centrifugation, ii/use of a more potent inhibitory anti-TF antibody iii/ use of FVII and a fluorogenic substrate to increase specificity.Results: The specificity of the MV TF activity assay was demonstrated by the absence of activity on MV derived from a knock-out-TF cell line using an anti-human TF monoclonal antibody called SBTF-1, which shows a higher TF inhibitory effect than the anti-human TF monoclonal antibody called HTF-1. Experiments using blood from healthy individuals, stimulated or not by LPS, or plasma spiked with 3 different levels of MV, demonstrated that the new assay was more sensitive and this allowed detection of MV TF activity in platelet free plasma (PFP) samples from healthy individuals. However, the assay was limited by an inter-assay variability, mainly due to the centrifugation step.Conclusions: We have improved the sensitivity of the MV TF activity assay without losing specificity. This new assay could be used to evaluate levels of TF-positive MV as a potential biomarker of thrombotic risk in patients