Fig. 6.

RIPK3 mediates the heightened IL-1β production, autoimmune responses, and EAE progression evoked by the loss of myeloid caspase-8. (A and B) Suppression of IL-1β production by RIPK1 inhibition. WT and C8KO^LysM BMDM were activated with LPS (10 ng/mL) with or without Nec1 (20 μM) for 24 h, and the level of IL-1β was measured in the supernatants. n = 5 per genotype. (B) Ripk3 deletion in C8KO^LysM BMDM completely abrogated LPS-induced production of mature IL-1β. IL-1β was measured in the supernatants at 24 h. C8KO^LysM, n = 8; C8KO^LysM/Ripk3^−/−, n = 4. (C) Nec1 inhibited MOG-induced IL-1β and IFN-γ production in C8KO^LysM BMDM/2D2 T cell cocultures. BMDM were cocultured with Th1 polarized 2D2 T cells in the absence and presence of MOG_35–55 and Nec1 (20 μM) for 24 h, and cytokines were measured in the supernatants. n = 3 per genotype. (D) Ripk3 deletion in C8KO^LysM BMDM abolished MOG-elicited IL-1β production and resultant production of GM-CSF, IFN-γ, and IL-17A by T cells. C8KO^LysM, n = 5; C8KO^LysM/Ripk3^−/−, n = 4; Ripk3^−/−, n = 3. (E) Mean clinical scores of mice subjected to MOG-induced EAE. C8KO^LysM, n = 7; C8KO^LysM/Ripk3^−/−, n = 8; WT, n = 20; Ripk3^−/−, n = 15. Two-way ANOVA with Bonferroni’s post hoc multiple test was used to assess statistical significance between groups. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. The significance is indicated between C8KO^LysM and C8KO^LysM/Ripk3^−/− and between WT controls and Ripk3^−/− mice.