Spatiotemporal Immune Inflammation modulates 3D NFκB signaling interactomics of multiprotein supercomplexes

The NFκB signaling pathway orchestrates inflammatory responses through the dynamic assembly and dissociation of membrane-proximal multiprotein supercomplexes, yet their spatiotemporal organization within the three-dimensional (3D) intracellular space has remained unresolved at single-cell resolution. Here, we present the first volumetric, in situ profiling of endogenous NFκB protein-protein interactions (PPIs) using iterative sequential proximity ligation assay (iseqPLA) combined with spinning disk confocal microscopy and 3D reconstruction. Across 01-3T3 mouse fibroblasts, IMR-90 human fibroblasts, and cystic fibrosis patient-derived macrophage cocultures, we characterize supercomplex dissociation kinetics, p65 nuclear translocation dynamics, and negative feedback engagement over a 105-minute cytokine time course. We demonstrate that 3D volumetric quantification resolves PPI distributions obscured by conventional 2D maximum intensity projections, that extracellular matrix coating critically determines the fraction of NFκB-responsive cells, and that CF airway-conditioned macrophages amplify paracrine NFκB signaling in adjacent fibroblasts. A transfer learning-based scGPT foundation model, trained on curated in vitro and in vivo transcriptomic datasets, confirms statistically significant enrichment of our selected NFκB gene panel within inflammation-relevant transcriptional feature space. This work establishes a generalizable 3D spatial interactomics framework for dissecting signaling supercomplex dynamics in health and disease.