Flavin adenine dinucleotide is an endogenous suppressor for cytosolic DNA and RNA sensors to modulate innate immunity.

Cytosolic DNA and RNA sensing is crucial for innate immunity, playing essential roles in pathogen defense and autoinflammation induction. We reported the endogenous metabolite flavin adenine dinucleotide (FAD) as a molecular brake restraining both cytosolic DNA and RNA sensing. It bound directly to cytosolic nucleic acid sensors cyclic GMP-AMP synthase (cGAS) and retinoic acid-inducible gene I (RIG-I), occupying catalytic pockets to suppress their activity and downstream immune responses. Physiologically, FAD prevented self-nucleic acid-induced sterile inflammation and maintained immune homeostasis. FAD deficiency due to FAD synthase (FLAD1) ablation exacerbated auto-inflammation and cellular senescence. Upon viral infection, reduced FLAD1 activity lowered FAD amounts, which removed its inhibitory control over DNA and RNA sensors, thus facilitating extensive interferon-I (IFN-I) signaling activation. Consequently, FLAD1 depletion strengthened the innate immune response and protected mice from viral infection. Our findings identify FAD as a natural suppressor of both cytosolic DNA and RNA sensing, offering therapeutic potential for inflammatory diseases.