Real-Time Single-Cell Kinetics of Innate Immune Activation 2264282

Abstract Introduction The innate immune system depends on a network of signaling pathways to detect and respond to viral infections, yet the degree of interdependence between pathways remains poorly understood. In this study, we investigate whether key innate immune pathways activate in a coordinated or independent manner during viral sensing. Methods Using real-time live-cell imaging of fluorescently tagged signaling proteins, in combination with western blotting, immunofluorescence, and single-molecule RNA FISH, we track the activation dynamics of individual pathways at single-cell resolution in response to diverse viral stimuli. Results Our results reveal that innate immune pathways are activated independently and maintain functionality in the absence of parallel pathways. IRF3, PKR, and RNase L innate immune pathways all retain full capacity to activate independently of each other. Conclusion This finding stands in contrast to prevailing models that propose extensive crosstalk and dependence. These results suggest that innate immune pathways are evolutionarily optimized to operate autonomously, providing a robust antiviral response during conditions of pathway inhibition or disruption. This work challenges assumptions in the field and redefines our understanding of innate immune signaling architecture. Funding Source NIH Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)