Fig. 5

HeVc peptide-induces maturation of IL-1β and caspase-1 in human THP-1 macrophages and PBMCs. Human THP-1 cells seeded in a 12-well plate (5 × 10⁵ cells per well) were differentiated with 4 µM PMA in media for 16 h before their media was changed and incubated for a further 48 h to differentiate into macrophages. THP-1 derived macrophages were primed with 100 ng/ml LPS-B5 for 3 h in the presence or not of B) MCC950 and A-B) stimulated with 200 µg/ml of HeVc peptide for 6 h. Supernatants were harvested for the detection of IL-1β secretion via ELISA. C-D) THP-1 macrophages were treated with MCC950 or not where indicated and challenged with aggregated HeVc peptide or silica for 6 h respectively. Supernatants were harvested and proteins separated on a 4–12% SDS-PAGE gel before visualization of IL-1β and caspase-1 was conducted by immunoblot. E) hPBMCs from 4 donors were separated from 30 ml blood via density-gradient separation and seeded into a 96-well plate (2.5 × 10⁵ cells per well). hPBMCs were primed for 3 h with 50 pg/ml LPS in the presence or not of MCC950 before stimulation with HeVc peptide (200 µg/ml) or Scramble (200 µg/ml) for 6 h. Supernatant was analysed via ELISA for IL-1β secretion. A-B) Results are representative of 3 independent experiments conducted in triplicate and are presented as mean ± SD. E) Results are the mean ± SEM of four independent donors. Ordinary One-way ANOVA with Dunnett’s test for multiple comparisons *p < 0.05, **p < 0.01, ***p < 0.001