Figure 1

Extracellular NLRP3-YFP inflammasome particles are internalized by primary human coronary artery smooth muscle cells (HCASMC) and induce caspase-1 and IL-1β activation. (A) Schematic overview of study objective. Constitutively active mutant HEK NLRP3-YFP (p.D303N) cells were used for isolation of cell-free NLRP3-YFP inflammasome particles that are used for treatment of HCASMC (scale bar: 10 µm). (B) Internalization of extracellular NLRP3-YFP inflammasomes in HCASMC after 4 h of incubation determined by immunofluorescent staining (N = 3) with an anti-YFP antibody or IgG isotype control and fluorescently-labeled anti-rabbit Alexa 647 secondary antibody (scale bar: 50 µm). Alexa Fluor 555 Phalloidin and DAPI were used for F-Actin and nucleus staining, respectively. Z-stacks with xz and yz focal planes (white dashed lines) showing internalized NLRP3-YFP inflammasome in HCASMC. (C) Internalization of extracellular NLRP3-YFP inflammasomes (purified particles) (yellow). HCASMC were incubated for 4 h. Uptake was confirmed via confocal microscopy. Plasma membrane staining (red) was carried out using Cell Mask Deep Red (scale bar: 20 µm). Micrographs depicting internalization and subcellular localisation of NLRP3-YFP inflammasome particles are shown in overview and enhanced magnification (scale bar: 5 µm). (D) For quantification, four representative fields of view were analyzed. (E) Representative ImageStream analysis of HCASMC showing internalized extracellular NLRP3-YFP inflammasome (scale bar: 10 µm) by merging the brightfield image (BF), YFP signal of the NLRP3-YFP inflammasome and the immunostained signal of the anti-YFP/ anti-rabbit Alexa 647 antibody. (F-K) HCASMC were treated with extracellular NLRP3-YFP inflammasome particles (3:1) for 4 h and 24 h with our without pre-incubation of Cytochalasin D (CytoD, 4 µM) for 30 min and total protein lysate was used for immunoblotting. Immunoblot (N = 4) of (F) NLRP3, pro-IL-1β and mature IL-1β 17 kDa, pro-caspase-1 and activated caspase-1 p20 (4 h) as well as endogenous NLRP3 and ASC after 24 h. The corresponding densitometric analysis of (G) mature cleaved IL-1β and (H) activated caspase-1 p20 as well as (I) IL-1β secretion (pg/ml) is shown (after 4 h). As positive control for endogenous inflammasome activation and IL-1β secretion, cells were stimulated with LPS (1 µg/ml, 3 h) and Nigericin (10 µM, 90 min). (J, K) Densitometric analysis of NLRP3 and ASC protein level after 24 h of stimulation with extracellular NLRP3-YFP inflammasome particles (N = 4). β-Actin was used as housekeeping control. To exclude effects of particle isolation, same protocol for inflammasome particle isolation was performed on WT HEK cells and were used for treatment (WT con) of HCASMC. Data were normalized on β-Actin and set at 1. (L) Membrane disruption was measured by the release of lactate dehydrogenase (LDH) into supernatant of HCASMC treated with NLRP3-YFP inflammasome particles (3:1 particles/cell) for 4 h. LDH values were normalized to positive LDH control which was set 100%. Immunoblot (N = 3) of (M) Gasdermin D (full-length 53 kDa) and its active cleaved N-terminal fragment (30 kDa). β-Actin was used as housekeeping control. (N) Densitometric analysis of the cleaved Gasdermin D fragment (30 kDa) was performed and normalized on untreated control which was set at 1. HCASMC treated with LPS + Nig were used as positive control. Differences between the groups were analysed by One-way ANOVA and uncorrected Fishers LSD post-hoc test (*p < 0.05).