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

Reduction of mortality, cardiac damage, and cerebral damage by IL-1 inhibition in a murine model of TTP

International audienceAbstract Thrombotic thrombocytopenic purpura (TTP), a rare but fatal disease if untreated, is due to alteration in von Willebrand factor cleavage resulting in capillary microthrombus formation and ischemic organ damage. Interleukin-1 (IL-1) has been shown to drive sterile inflammation after ischemia and could play an essential contribution to postischemic organ damage in TTP. Our objectives were to evaluate IL-1 involvement during TTP and to test the efficacy of the recombinant IL-1 receptor antagonist, anakinra, in a murine TTP model. We retrospectively measured plasma IL-1 concentrations in patients with TTP and controls. Patients with TTP exhibited elevated plasma IL-1α and -1β concentrations, which correlated with disease course and survival. In a mouse model of TTP, we administered anakinra (IL-1 inhibitor) or placebo for 5 days and evaluated the efficacy of this treatment. Anakinra significantly reduced mortality of mice (P < .001). Anakinra significantly decreased TTP-induced cardiac damage as assessed by blood troponin concentrations, evaluation of left ventricular function by echocardiography, [18F]fluorodeoxyglucose positron emission tomography of myocardial glucose metabolism, and cardiac histology. Anakinra also significantly reduced brain TTP-induced damage evaluated through blood PS100b concentrations, nuclear imaging, and histology. We finally showed that IL-1α and -1β trigger endothelial degranulation in vitro, leading to the release of von Willebrand factor. In conclusion, anakinra significantly reduced TTP mortality in a preclinical model of the disease by inhibiting both endothelial degranulation and postischemic inflammation, supporting further evaluations in humans

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

International audienceObjective IL-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. Methods MVs 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. Results THP-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. Conclusion MVs 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