Dissecting temporal regulation of the TLR4–IL-6 axis using systematic targeted inhibition

Acute systemic inflammatory disorders, such as cytokine release syndrome or sepsis, remain among the leading causes of mortality. To better understand early inflammatory dynamics, we developed an ex vivo model using human peripheral blood mononuclear cells to study IL-6 release kinetics via the Toll-like receptor 4 pathway. A consistent kinetic profile was observed and described with a mixed-effects model. To further dissect the signaling hierarchy, we investigated the response dynamics of multiple inhibitors targeting the various stages of signaling at different timepoints. The results suggest individual differences with common patterns of IL-6 kinetics and indicate that sustained IL-6 release requires continuous upstream signaling. Furthermore, important feedback mechanisms were identified, with NF-κB and p38 emerging as critical regulators when compared with other members. The translational relevance of our model was further supported and validated by the use of clinically applied drugs, which demonstrated consistent effects with their known clinical activity.