Caspase-1-Dependent Pyroptosis Mediates Adjuvant Activity of Platycodin D as an Adjuvant for Intramuscular Vaccines

Platycodin D (PD) is a potent adjuvant with dual Th1 and Th2 potentiating activity, but its mechanisms of action remain unclear. Here, the C2C12 myoblast cell line and mice were used as in vitro and in vivo models to identify potential signaling pathways involved in the adjuvant activity of PD. PD induced a transient cytotoxicity and inflammatory response in the C2C12 cells and in mouse quadricep muscles. A comparative analysis of microarray data revealed that PD induced similar gene expression profiles in the C2C12 cells and in the quadricep muscles, and triggered rapid regulation of death, immune, and inflammation-related genes, both in vivo and in vitro. It was further demonstrated that caspase-1-dependent pyroptosis was involved in the PD-induced cytotoxicity and inflammatory response in the C2C12 cells via the Ca²⁺-c-jun N-terminal kinase (JNK)/p38 mitogen-activated protein kinase (MAPK)-NLR family pyrin domain containing 3 (NLRP3) inflammasome signaling pathway. Consistently, the in vivo analysis revealed that a local blockage of NLRP3 and caspase-1 inhibited PD-induced cytokine production and immune cell recruitment at the injection site, and impaired the adjuvant activity of PD on antigen-specific immune responses to model antigen ovalbumin (OVA) in mice. These findings identified the caspase-1-dependent adjuvanticity of PD and expanded the current knowledge on the mechanisms of action of saponin-based adjuvants.