Toll-like receptor 4-mediated inflammation triggered by extracellular IFI16 is enhanced by lipopolysaccharide binding

Author summary IFI16 is a nuclear protein involved in a variety of physiological processes, including cell cycle regulation, tumor suppression, and virus sensing. Emerging evidence indicates that IFI16 is released in the extracellular milieu under injury or stress conditions. Here we show that extracellular IFI16 acts as a damage-associated molecular pattern (DAMP), triggering inflammation through Toll-like receptor 4 (TLR4) activation. Furthermore, we demonstrate that IFI16 activity is potentiated upon binding to subtoxic concentrations of strong TLR4-activating lipopolysaccharide (LPS) variants, which are known to be present in various pathological settings other than gram-negative infections. Our study provides new insights into the role of extracellular IFI16 during low-grade endotoxemia. Damage-associated molecular patterns (DAMPs) are endogenous molecules activating the immune system upon release from injured cells. Here we show that the IFI16 protein, once freely released in the extracellular milieu of chronically inflamed tissues, can function as a DAMP either alone or upon binding to lipopolysaccharide (LPS). Specifically, using pull-down and saturation binding experiments, we show that IFI16 binds with high affinity to the lipid A moiety of LPS. Remarkably, IFI16 DAMP activity is potentiated upon binding to subtoxic concentrations of strong TLR4-activating LPS variants, as judged by TLR4-MD2/TIRAP/MyD88-dependent IL-6, IL-8 and TNF-alpha transcriptional activation and release in stimulated monocytes and renal cells. Consistently, using co-immunoprecipitation (co-IP) and surface plasmon resonance (SPR) approaches, we show that IFI16 is a specific TLR4-ligand and that IFI16/LPS complexes display a faster stimulation turnover on TLR4 than LPS alone. Altogether, our findings point to a novel pathomechanism of inflammation involving the formation of multiple complexes between extracellular IFI16 and subtoxic doses of LPS variants, which then signal through TLR4.