Fig. 3.

Nlrp3 is not required for RBC alloimmunization. a) Theoretical model of NLRP3 inflammasome activation by stored RBC DAMPs in a dendritic cell. Following recognition of possible inflammatory stimuli, such as ATP, iron or heme, NLRP3 oligomerizes, recruits ASC and caspase-1, which enzymatically cleaves pro-cytokines, resulting in secretion of active IL-18 and IL-1β. MHC-II is upregulated following activation and presents RBC antigens to antigen-specific T cells. ASC, apoptosis-associated speck-like protein containing a CARD. b,c) Inflammatory cytokines (b) and anti-RBC alloantibodies (c) in sera of indicated mice following transfusion with stored HOD RBCs, quantified as in Fig. 1. n = 3–5 mice/group. Dotted line in (b) indicates level of serum cytokines in naïve mice from the same experiment. Data are representative of 5 independent experiments. d) IL-1β, measured by ELISA, in supernatants of ex vivo cultured thioglycollate-elicited peritoneal macrophages from indicated mice primed with 50 ng/mL LPS for 16–18 h prior to stimulation with either 500 ng/mL alum or 5 mM ATP for 8 h. Dotted line indicates level of detection of ELISA. Mean with s.d. from triplicate wells. Representative data from 3 independent experiments. NS, not significant; *p < 0.05; ****p < 0.0001.