Fig. 5: [Ca²⁺]_exmediated calciprotein particle uptake mediates inflammasome activation.

a–h Detection of IL-1β via ELISA in cell culture supernatants of human freshly isolated peripheral blood monocytes from the indicated number of donors after 16 h of incubation with LPS (100 ng/ml) and the indicated substances. a Treatment with different concentrations of CPPs and the indicated [Ca²⁺] in RPMI1640/10%FBS containing 1 mM [P_i]. b Time course of IL-1β release after addition of 2.5 mM [Ca²⁺] to monocytes in 1 mM [P_i] plus CPPs compared to monocytes in RPMI1640/10%FBS containing 5.6 mM [P_i]. c Pre-incubation with 1 µM Cytochalasin D (Cyto D) or DMSO prior to stimulation with 2.5 mM [Ca²⁺] or 3 mM ATP or 100 µg/ml MSU in RPMI1640/10%FBS containing 5.6 mM [P_i]. d Pre-incubation with 3 µM CA-074-Me or DMSO for 30 min prior to stimulation with 2.5 mM added [Ca²⁺] or 3 mM ATP or 100 µg/ml MSU in RPMI1640/10%FBS containing 5.6 mM [P_i]. e Incubation in filtrated (100 nm) or non-filtrated (no filter) RPMI1640/10%FBS with 2.5 mM added [Ca²⁺]. Medium was either pre-incubated with [Ca²⁺] for 120 min prior to filtration (t = 120 min) or directly filtrated (t = 0) through a 100 nm pore-size filter. Visualization of particle reduction after filtration is shown in the graph inset, particulate matter was pelleted by 2 h centrifugation at 16,000 × g. f Incubation in RPMI1640/10%FBS containing 5.6 mM [P_i] with additional fetuin-A concentrations and stimulation with 2.5 mM added [Ca²⁺]. g Addition of phosphonoformic acid (PFA) into RPMI1640/10%FBS containing 5.6 mM [P_i] prior to stimulation with 2.5 mM added [Ca²⁺] or 3 mM ATP or 100 µg/ml MSU. h Treatment of monocytes with the indicated concentrations of albumin-coated BaSO₄ nanoparticles in the absence (ctrl) or presence of 2.5 mM added [Ca²⁺] or 3 mM ATP or 100 µg/ml MSU. a–h Box-and-Whisker plots show median, 25–75th percentile, and min/max whiskers, bar charts show mean ± s.e.m. Statistical analysis was performed using two-tailed Mann–Whitney U test (a, d, e, g) or two-tailed t-test b, c. p-values are indicated as *p < 0.05, **p < 0.01, ***p < 0.001. i Correlation between IL-1β release (16 h) and macropinocytosis (calcein uptake after 45 min) of monocytes from 10 healthy donors after stimulation with LPS and 2.5 mM added [Ca²⁺] in RPMI1640/10%FBS containing 5.6 mM [P_i]. Two-tailed Pearson correlation coefficient and level of significance as indicated.