Figure 5: IL-21-induced IL-1β by cDCs is independent of the canonical caspase-containing inflammasomes.

(a) cDCs were rested 1 h, treated with 100 ng ml⁻¹ IL-21 or LPS at the indicated time points, and intracellular pro-IL-1β expression was determined. β-actin was used as control. Shown is one of two similar experiments. (b) cDCs were treated as in a, with 5 mM ATP added 1 h prior indicated time points in LPS-stimulated samples, and the secretion of IL-1β was determined by enzyme-linked immunosorbent assay (ELISA). Shown are combined results of two independent experiments; error bars are means±s.e.m. (c) CD4⁺ T cells from WT or Il1r^−/− mice were cultured in Th17 cell differentiation conditions for 2 days, then supernatant from a 24 h, IL-21-treated cDC culture was added to the Th17 cells and incubated for 2 days, with or without addition of 10 μg ml⁻¹of anti-IL-1β. Expression of IL-2Rα (MFI) was determined by flow cytometry. The amount of biologically active IL-1β was determined using a standard curve constructed by assaying recombinant IL-1β. Shown are the combined results of two independent experiments with total of six samples. (d,e) WT, Casp1^−/−, Nlrp3^−/− and Pycard^−/− cDCs were rested 1 h. In d, cDCs were then treated with 100 ng ml⁻¹ LPS for 20–24 h with 5 mM ATP added in the final 1 h, and IL-1β assessed. Data are from two experiments; error bars are means±s.e.m. In e, cDCs were then treated with IL-21 for 20–24 h and IL-1β protein determined. Data are from five experiments. P values of IL-21-treated WT samples as compared with Casp1^−/−, Nlrp3^−/− and Pycard^−/− samples are 0.99, 0.22 and 0.96, respectively; error bars are means±s.e.m. (f) cDCs from Ripk3^−/−, Ripk3^+/−Casp8^+/− and Ripk3^−/−Casp8^−/− mice were treated as in e, and IL-1β assessed. Data shown are from three experiments. P values of IL-21-treated Ripk3^−/− sample compared with Ripk3^+/−Casp8^+/− and Ripk3^−/−Casp8^−/− samples are 0.57 and 0.93, respectively. In b and d–f, IL-1β production in the culture supernatant was determined by ELISA. Pro-IL-1β induced by IL-21 in the culture supernatant was minimal, based on a pro-IL-1β-specific ELISA. Statistical analysis was performed by Student’s t-test.