Extracellular NLRP3 inflammasome particles are internalized by human coronary artery smooth muscle cells and induce pro-atherogenic effects

Abstract

Inflammation driven by intracellular activation of the NLRP3 inflammasome is involved in the pathogenesis of a variety of diseases including vascular pathologies. Inflammasome specks are released into the extracellular compartment from disrupting pyroptotic cells. The potential uptake and function of extracellular NLRP3 inflammasomes in human coronary artery smooth muscle cells (HCASMC) are unknown. Fluorescently labeled NLRP3 inflammasome particles were isolated from a mutant NLRP3-YFP cell line and used to treat primary HCASMC for 4 and 24 h. Fluorescent and expressional analyses showed that extracellular NLRP3-YFP particles are internalized into HCASMC, where they remain active and stimulate intracellular caspase-1 (1.9-fold) and IL-1β (1.5-fold) activation without inducing pyroptotic cell death. Transcriptomic analysis revealed increased expression level of pro-inflammatory adhesion molecules (ICAM1, CADM1), NLRP3 and genes involved in cytoskleleton organization. The NLRP3-YFP particle-induced gene expression was not dependent on NLRP3 and caspase-1 activation. Instead, the effects were partly abrogated by blocking NFκB activation. Genes, upregulated by extracellular NLRP3 were validated in human carotid artery atheromatous plaques. Extracellular NLRP3-YFP inflammasome particles promoted the secretion of pro-atherogenic and inflammatory cytokines such as CCL2/MCP1, CXCL1 and IL-17E, and increased HCASMC migration (1.8-fold) and extracellular matrix production, such as fibronectin (5.8-fold) which was dependent on NFκB and NLRP3 activation. Extracellular NLRP3 inflammasome particles are internalized into human coronary artery smooth muscle cells where they induce pro-inflammatory and pro-atherogenic effects representing a novel mechanism of cell-cell communication and perpetuation of inflammation in atherosclerosis. Therefore, extracellular NLRP3 inflammasomes may be useful to improve the diagnosis of inflammatory diseases and the development of novel anti-inflammatory therapeutic strategies.

Walking down Skeletal Muscle Lane: From Inflammasome to Disease

“…In contrast to other types of cell death, such as necrosis and apoptosis, pyroptosis causes the rupture of the cell membrane via pores formation and induces the release of mature inflammatory cytokines, damage associated molecular patterns (DAMPs) and NLRP3 inflammasome specks into the extracellular compartment where they remain active, ultimately resulting in an amplification of the inflammatory response [ 20 , 21 , 22 ]. Pyroptosis may be a physiologic process during the acute inflammatory response by maintaining cell homeostasis and preventing excessive cell proliferation, but under chronic disease conditions, it will lead to uncontrolled inflammation, excessive cell death and tissue remodeling [ 23 , 24 ].…”

Extracellular Inflammasome Particles Are Released After Marathon Running and Induce Proinflammatory Effects in Endothelial Cells

“…These data identify extracellular inflammasome particles as a mechanism that mediates inflammation from the site of damaged tissue to the endothelium. Previous findings of our group demonstrated that extracellular NLRP3 particles are also ingested by human coronary artery smooth muscle cells and macrophages where they induce pro-inflammatory and atherogenic signaling by increasing the extracellular matrix production, promoting the secretion of pro-atherogenic and inflammatory cytokines and cell migration ( Gaul et al, 2021 ).…”

“…Extensive exercise with a high mechanical muscular load can mediate systemic inflammation via the release of extracellular inflammasome complexes. We and others propose that these pyroptosis-derived NLRP3 inflammasome particles exert pro-atherogenic effects ( Gaul et al, 2021 ; Sun et al, 2021 ).…”

“…HCAEC were treated with NLRP3-YFP particles for 24 h. Cells were fixed with 4% paraformaldehyde, permeabilized and incubated with an anti-YFP (GFP) antibody (#6556, Abcam), anti-YFP Alexa555 antibody (A-31851, Invitrogen), anti-ICAM-1 (#MA5407, Invitrogen), IgG isotype control (#RIgG, #MigG, Dianova), goat anti-mouse Cy3 (#115-165-146, Dianova), donkey anti-rabbit Alexa-488 (A-21206, Invitrogen), or goat anti-rabbit Alexa-647 secondary antibody (A-21245, Invitrogen) to analyze the internalization of extracellular NLRP3-YFP oligomers or ICAM-1 expression. DAPI, Hoechst, and F-actin Phalloidin Alexa 555/ Alexa647 were used for nucleus and F-actin staining, respectively ( Gaul et al, 2021 ). Cells were visualized on a ZEISS Axiovert 200 M fluorescent microscope.…”

“…Upon activation, NLRP3 nucleates with apoptosis-associated speck-like protein (ASC) to form an ASC speck, leading to caspase-1-mediated proteolytic activation of interleukin-1β (IL-1β) and interleukin-18 (IL-18), and the induction of inflammatory, pyroptotic cell death. Recent evidence shows that pyroptotic cells can subsequently release ASC specks into the extracellular space where they may perpetuate inflammation ( Gaul et al, 2021 ). Extracellular ASC specks have been described in chronic obstructive pulmonary disease, septic patients, and myelodysplastic syndrome ( Franklin et al, 2014 ; Basiorka et al, 2018 ; Martínez-García et al, 2019 ).…”