RIPK3-dependent sequential recruitment of MLKL and RIPK1 drives PANoptotic cell death and chemokine production

PANoptosis, an immunogenic programmed cell death (PCD) modality, integrates features of pyroptosis, apoptosis, and necroptosis through assembly of the PANoptosome complex. Despite its conceptualization as a distinct PCD form, PANoptosis remains controversial due to insufficient characterization of its morphological hallmarks and molecular regulation. This study aimed to investigate the molecular mechanisms underlying the assembly of PANoptosomes in the PANoptotic pathway. We identified a novel receptor-interacting protein kinase 3 (RIPK3)-initiated PANoptotic pathway that functions without pattern recognition receptors (PRRs) and the ASC (apoptosis-associated speck-like protein containing a CARD) inflammasome. Using multimodal imaging and biochemical approaches, we identify unique morphological signatures distinguishing PANoptotic cells from canonical pyroptotic, apoptotic, or necroptotic counterparts. Mechanistically, RIPK3 forms round homopolymeric scaffolds—distinct from necroptotic amyloid-like fibers—to sequentially recruit MLKL and RIPK1, forming a dynamic RIPK3-MLKL-RIPK1-FADD-caspase-8 complex (RIPK3-PANoptosome). This platform coordinates concurrent activation of pyroptotic, apoptotic, and necroptotic effectors. Cross-regulatory interactions between these pathways establish a homeostatic system where perturbations bias death modality into a certain cell death type, altering the death process and outcomes. Functionally, PANoptotic cells orchestrate chemokine secretion through parallel kinase-dependent (RIPK3-MLKL) and kinase-independent (RIPK1-IKK-NF-κB) mechanisms, driving macrophage recruitment. Our findings resolve the molecular logic of PANoptosome assembly, redefine PANoptosis as a tunable PCD paradigm, and establish its role in immunomodulation, providing a framework for targeting inflammatory cell death in disease.