A live-cell reporter to track Drosophila innate immunity reveals heterogeneity in NF-κB activation and target gene expression
OA Version
Citation
Abstract
Conserved NF-κB signaling pathways are critical for shaping innate immune responses across species. Yet, we lack a complete understanding of how pathway elements generate diverse responses to varying stimuli. Defining the origins and consequences of this heterogeneity can improve strategies for predicting and managing infection outcomes. Drosophila melanogaster is a powerful model organism for studying innate immunity due to its conservation of key signaling pathways and lack of adaptive immune response masking. In this dissertation, I develop and use live Drosophila S2* reporter cells to investigate the spatiotemporal dynamics of the IMD innate immune pathway, which responds to Gram-negative bacterial cues and activates the NF-κB transcription factor Relish. I engineer single-cell reporters that simultaneously track Relish and transcription of antimicrobial peptide (AMP) genes under its control. Using this system, I discover distinct categories of Relish spatiotemporal dynamics, governed in part by pre-stimulus cell states, and show that these behaviors predict transcriptional outcomes. I further demonstrate that AMP gene enhancer architecture, particularly the number of κB-binding motifs, tunes transcriptional activity, and that AMP expression exhibits gene-specific burst kinetics. Additional experiments rule out oscillatory signaling and rapid protein turnover as sources of heterogeneity, instead implicating negative feedback regulation. Together, this work reveals that NF-κB signaling and downstream transcription in Drosophila innate immunity are both heterogeneous and tunable. These findings establish a new framework for studying innate immune regulation at single-cell resolution and open avenues for cross-species comparisons of immune strategies.
Description
2025