Netosis and Inflammasomes in Large Vessel Occlusion Thrombi

Abstract

The inflammatory response appears to play a critical role in clotting in which neutrophil extracellular traps (NETs) are the major drivers of thrombosis in acute ischemic stroke (AIS). The inflammasome is an innate immune complex involved in the activation of interleukin (IL)-18 and IL-1β through caspase-1, but whether the inflammasome plays a role in NETosis in AIS remains poorly understood. Here we assessed the levels of inflammasome signaling proteins in NETs and their association with clinical and procedural outcomes of mechanical thrombectomy for AIS. Electron microscopy and immunofluorescence indicate the presence of NETs in thrombi of patients with AIS. Moreover, the inflammasome signaling proteins caspase-1 and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) were also present in clots associated with the marker of NETosis citrullinated histone 3H (CitH3). Analysis of protein levels by a simple plex assay show that caspase-1, ASC and interleukin (IL)-1β were significantly elevated in clots when compared to plasma of AIS patients and healthy controls, while IL-18 levels were lower. Moreover, multivariate analyses show that IL-1β levels in clots contribute to the number of passes to achieve complete recanalization, and that ASC, caspase-1 and IL-18 are significant contributors to time to recanalization. Thus, inflammasome proteins are elevated in NETs present in thrombi of patients with AIS that contribute to poor outcomes following stroke.

Inflammasome activation in neutrophils of patients with severe COVID-19

“…Indeed, we showed that speck formation occurs transiently in murine neutrophils before nuclear decondensation. Our results are reinforced by recent studies suggesting a link between NETs and the presence of extracellular inflammasomes 42,43 . Chen et al showed via electron microscopy and immunofluorescence that inflammasome signaling proteins like caspase-1 and ASC speck are present in NETs of human thrombi and associated with H3cit, the marker of NETosis 43 .…”

Clot Composition Analysis as a Diagnostic Tool to Gain Insight into Ischemic Stroke Etiology: A Systematic Review

“…66,93 Neutrophil extracellular traps (NETs) have been identified as part of the clot scaffold by using antibodies against NETosis biomarkers (citrullinated histones) and histochemical staining of extracellular DNA. 63,65,66,76,[93][94][95]99 NETs were visualized in almost all (79.1% to 100%) of the analyzed thrombi in different studies, 63,65,66,76,93,95 in amounts ranging Values are presented as weighted mean±pooled standard deviation or number. PLT, platelet; FBR, fibrin; RBC, red blood cell; WBC, white blood cell.…”

“…76 NET signals were observed as being confined within cells, filopodia-like structures, or web-like structures, 65 especially in the outer thrombus layers, 63,95 and almost exclusively within FBR-rich areas. 76 Inside the NETs, citrullinated histones were co-localized with inflammasome proteins (caspase-1 and apoptosis-associated speck-like protein containing a caspase-recruitment domain [ASC]), 99 granular neutrophil proteins (MPO) and extracellular DNA released from neutrophils. 93,95 The addition of histone-DNA complexes to FBR resulted in thicker fibers accompanied by increased rigidity, which contributed to the structural complexity and stabilization of the thrombi.…”

“…63,107 Other ultrastructural studies showed morphologic features consistent with the presence of NETs, calcified deposits and cholesterol crystals in the clots. 44,99…”

“…Moreover, the presence of nucleotide-binding oligomerization domain (NOD)-like receptor protein-1 and absent in melanoma-2, two receptors that interact with caspase-1 and ASC to form an inflammasome complex, was shown by immunoblot analysis. 99 Proteomic analysis has been used to characterize the protein cargo of thrombi (thousands) and commonly present proteins (hundreds). Functional bioinformatics analyses revealed protein clusters related to inflammation; 113 immunological functions, blood cell dependent functions, and peripheral vascular processes; 114 metabolic processes, inflammatory track, and cell proliferation, activation, or motility; 115 primary hemostasis, integrin and kinase signaling linked to integrins, glycolysis, and acute phase reactants.…”