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Interleukin-1? Mediates Arterial Thrombus Formation via NET-Associated Tissue Factor.

ABSTRACT: CANTOS reported reduced secondary atherothrombotic events in patients with residual inflammatory risk treated with the inhibitory anti-IL-1? antibody, Canakinumab. Yet, mechanisms that underlie this benefit remain elusive. Recent work has implicated formation of neutrophil extracellular traps (NETosis) in arterial thrombosis. Hence, the present study explored the potential link between IL-1?, NETs, and tissue factor (TF)-the key trigger of the coagulation cascade-in atherothrombosis. To this end, ST-elevation myocardial infarction (STEMI) patients from the Swiss multicenter trial SPUM-ACS were retrospectively and randomly selected based on their CRP levels. In particular, 33 patients with STEMI and high C-reactive protein (CRP) levels (? 10 mg/L) and, 33 with STEMI and low CRP levels (? 4 mg/L) were investigated. High CRP patients displayed elevated circulating IL-1?, NETosis, and NET-associated TF plasma levels compared with low CRP ones. Additionally, analysis of patients stratified by circulating IL-1? levels yielded similar results. Moreover, NETosis and NET-associated TF plasma levels correlated positively in the whole population. In addition to the above, translational research experiments provided mechanistic confirmation for the clinical data identifying IL-1? as the initial trigger for the release of the pro-coagulant, NET-associated TF. In conclusion, blunted TF presentation by activated neutrophils undergoing NETosis may provide a mechanistic explanation to reduced secondary atherothrombotic events as observed in canakinumab-treated patients in CANTOS.

SUBMITTER: Liberale L

PROVIDER: S-EPMC6947515 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Interleukin-1β Mediates Arterial Thrombus Formation via NET-Associated Tissue Factor.

Liberale Luca L Holy Erik W EW Akhmedov Alexander A Bonetti Nicole R NR Nietlispach Fabian F Matter Christian M CM Mach François F Montecucco Fabrizio F Beer Jürg H JH Paneni Francesco F Ruschitzka Frank F Libby Peter P Lüscher Thomas F TF Camici Giovanni G GG

Journal of clinical medicine 20191126 12

CANTOS reported reduced secondary atherothrombotic events in patients with residual inflammatory risk treated with the inhibitory anti-IL-1β antibody, Canakinumab. Yet, mechanisms that underlie this benefit remain elusive. Recent work has implicated formation of neutrophil extracellular traps (NETosis) in arterial thrombosis. Hence, the present study explored the potential link between IL-1β, NETs, and tissue factor (TF)-the key trigger of the coagulation cascade-in atherothrombosis. To this end ...[more]

PMID: 31779200

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Project description:Interleukin-1β (IL-1β), a major pro-inflammatory cytokine, is a leaderless cytosolic protein whose secretion does not follow the classical endoplasmic reticulum-to-Golgi pathway, and for which a canonical mechanism of secretion remains to be established. Neutrophils are essential players against bacterial and fungi infections. These cells are rapidly and massively recruited from the circulation into infected tissues and, beyond of displaying an impressive arsenal of toxic weapons effective to kill pathogens, are also an important source of IL-1β in infectious conditions. Here, we analyzed if an unconventional secretory autophagy mechanism is involved in the exportation of IL-1β by these cells. Our findings indicated that inhibition of autophagy with 3-methyladenine and Wortmannin markedly reduced IL-1β secretion induced by LPS + ATP, as did the disruption of the autophagic flux with Bafilomycin A1 and E64d. These compounds did not noticeable affect neutrophil viability ruling out that the effects on IL-1β secretion were due to cell death. Furthermore, VPS34IN-1, a specific autophagy inhibitor, was still able to reduce IL-1β secretion when added after it was synthesized. Moreover, siRNA-mediated knockdown of ATG5 markedly reduced IL-1β secretion in neutrophil-differentiated PLB985 cells. Upon LPS + ATP stimulation, IL-1β was incorporated to an autophagic compartment, as was revealed by its colocalization with LC3B by confocal microscopy. Overlapping of IL-1β-LC3B in a vesicular compartment peaked before IL-1β increased in culture supernatants. On the other hand, stimulation of autophagy by cell starvation augmented the colocalization of IL-1β and LC3B and then promoted neutrophil IL-1β secretion. In addition, specific ELISAs indicated that although both IL-1β and pro-IL-1β are released to culture supernatants upon neutrophil stimulation, autophagy only promotes IL-1β secretion. Furthermore, the serine proteases inhibitor AEBSF reduced IL-1β secretion. Moreover, IL-1β could be also found colocalizing with elastase, suggesting both some vesicles containing IL-1β intersect azurophil granules content and that serine proteases also regulate IL-1β secretion. Altogether, our findings indicate that an unconventional autophagy-mediated secretory pathway mediates IL-1β secretion in human neutrophils.

Project description:Essentials Biological activity of human neutrophil peptide (HNP)-1 in hemostasis under physiological conditions is not fully understood. HNP-1 inhibits the adhesion/aggregation of murine platelets on a fibrillar collagen surface or an activated endothelial cell surface under flow. The anti-adhesion activity appears to depend on the terminal free thiols of HNP-1, which may inhibit VWF-VWF lateral associations. Our results suggest a protective role and potential novel therapeutic use of HNP-1 for arterial thrombosis. SUMMARY: Background Human neutrophil peptides (HNPs), also known as α-defensins, are released from degranulated neutrophils and play an important role in innate immunity. However, their biological roles in hemostasis under flow are not fully explored. Objective This study aims to determine the role of HNP-1 on platelet adhesion and aggregation on a collagen surface or ultra large von Willebrand factor (ULVWF) on endothelium under flow and elucidate the structural elements required for its activity. Methods Anticoagulated whole blood from wild-type or Adamts13-/- mice was incubated with a fluorescein-conjugated anti-human CD41 in the presence of increasing concentrations of a synthetic HNP-1 and perfused over a collagen surface or a tumor necrosis factor (TNF)-α activated murine endothelial cell surface under arterial flow. The rate of accumulation and the final surface coverage of fluoresceinated murine platelets or the rate of forming platelet-decorated ULVWF strings were determined using the BioFlux microfluidic system. Results HNP-1 inhibited the rate and final coverage of fluorescein-labeled murine platelets on a fibrillar collagen surface under flow (100 dyne/cm2 ) in a concentration-dependent manner and the anti-adhesive activity of HNP-1 depended on its terminal free cysteine thiols. HNP-1 (20 μM) also dramatically inhibited the formation of platelets-decorated ULVWF strings on TNF-α activated murine endothelial surface under arterial flow. Conclusions Our results demonstrate for the first time an antiplatelet adhesion or antithrombotic activity of HNP-1; this activity depends on its terminal free thiols, likely affecting VWF-VWF lateral associations. These findings may suggest a potential novel therapeutic strategy for arterial thrombosis.

Project description:Adipose tissue expansion during obesity is associated with increased macrophage infiltration. Macrophage-derived factors significantly alter adipocyte function, inducing inflammatory responses and decreasing insulin sensitivity. Identification of the major factors that mediate detrimental effects of macrophages on adipocytes may offer potential therapeutic targets. IL-1β, a proinflammatory cytokine, is suggested to be involved in the development of insulin resistance. This study investigated the role of IL-1β in macrophage-adipocyte cross-talk, which affects insulin signaling in human adipocytes. Using macrophage-conditioned (MC) medium and human primary adipocytes, we examined the effect of IL-1β antagonism on the insulin signaling pathway. Gene expression profile and protein abundance of insulin signaling molecules were determined, as was the production of proinflammatory cytokine/chemokines. We also examined whether IL-1β mediates MC medium-induced alteration in adipocyte lipid storage. MC medium and IL-1β significantly reduced gene expression and protein abundance of insulin signaling molecules, including insulin receptor substrate-1, phosphoinositide 3-kinase p85α, and glucose transporter 4 and phosphorylation of Akt. In contrast, the expression and release of the proinflammatory markers, including IL-6, IL-8, monocyte chemotactic protein-1, and chemokine (C-C motif) ligand 5 by adipocytes were markedly increased. These changes were significantly reduced by blocking IL-1β activity, its receptor binding, or its production by macrophages. MC medium-inhibited expression of the adipogenic factors and -stimulated lipolysis was also blunted with IL-1β neutralization. We conclude that IL-1β mediates, at least in part, the effect of macrophages on insulin signaling and proinflammatory response in human adipocytes. Blocking IL-1β could be beneficial for preventing obesity-associated insulin resistance and inflammation in human adipose tissue.

Dataset Information

Interleukin-1? Mediates Arterial Thrombus Formation via NET-Associated Tissue Factor.

Publications

Interleukin-1β Mediates Arterial Thrombus Formation via NET-Associated Tissue Factor.

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