Aernoud Fiolet

274 Chapter 11 RESULTS In vitro effects of NLRP3 inflammasome stimulation on EV NLRP3 protein levels In vitro stimulation of the NLRP3 inflammasome in THP-1 cells showed higher EV NLRP3 protein levels (10.4 ng/ml ± 1.3 ng/ml) compared to unstimulated controls (6.6 ng/ml ± 0.5 ng/ml) (difference 3.8 ng/ml, 95% CI 0.4 to 7.0; p=0.03) (Figure 1A). IL-1 β levels in medium were higher in stimulated THP-1 cells compared to unstimulated control cells (Supplemental Figure 1). In vitro stimulation of PMA- differentiated THP-1 cells showed higher EV NLRP3 protein levels (1.2 ng/ml ± 0.2) compared to unstimulated controls (0.36 ng/ml ± 0.02) (difference 0.84 ng/ ml, 95% CI 0.27 to 0.70; p<0.0001) (Figure 1B). Cell viability was >90% for all conditions, without significant differences between groups. Colchicine treatment in different dosages in combination with NLRP3 inflammasome stimulation significantly affected cell viability (<30%). Platelet stimulation did not result in EV NLRP3 protein release (data not shown). Figure 1. In vitro effects of NLRP3 inflammasome stimulation and colchicine treatment on EV NLRP3 protein levels. (A.) THP-1 cells were stimulated with LPS and ATP (~16 h). Stimulation led to a significant increase in EV NLRP3 protein release compared to unstimulated controls. (B.) THP-1 cells were differentiated to macrophages using PMA. PMA-differentiated THP-1 cells were stimulated with LPS and ATP (6h). Stimulation led to a significant increase in EV NLRP3 protein release. P values (*<0.05). Abbreviations: ATP, adenosine triphosphate; EV, extracellular vesicle; LPS, lipopolysaccharide; NLRP3, nucleotide-binding oligomerization domain-, leucine-rich repeat-, and pyrin domain-containing protein 3; PMA, phorbol 12-myristate 13-acetate