Quantitative Modeling of TLR Signaling Reveals Missing Negative Feedback Guiding Identification of TANK-IKKε Checkpoint.

Sensing of tissue injury or infection by Toll-like receptors (TLRs) must rapidly mobilize host defense, but that activation must also be terminated on an appropriate time scale to avoid sustained, tissue-damaging inflammation. The full network of molecular interactions that governs both the rapid onset and the properly timed shutoff of TLR signaling remains incompletely characterized at a deep mechanistic level. To this end, we built a rule-based model of mouse macrophage TLR4 signaling at the molecular-interaction level, parameterized with measured protein copy numbers, RNA-seq-based abundance estimates, literature- and structure-informed reaction rates, and 979 dynamic experimental constraints, achieving a level of granularity beyond that of prior TLR models. The trained model reproduced much of the TLR4-induced NF-κB and MAP kinase response, but it consistently failed to capture deactivation of MyD88, TRAF6-associated species, and IKKα/β. Rather than treating this as simple model error, we used the recurrent failure as a biological signal that localized missing regulation to the proximalMyD88-IRAK-TRAF6 module and motivated experimental evaluation of IKKε and its scaffold TANK. Loss of IKKε enhanced transcriptional, cytokine, MAP kinase, and NF-κB responses to MyD88-specific TLR ligands, and TANK deficiency produced a similar cellular phenotype while abolishing stimulus-induced IKKε phosphorylation. Deficiency of either protein increased IRAK1and TRAF6 ubiquitination without increasing MyD88 ubiquitination, placing the inhibitory checkpoint at or immediately downstream of the IRAK1-TRAF6 ubiquitin-signaling node. Overlapping but non-identical in vivo phenotypes further supported a shared regulatory axis with additional protein-specific functions. Together, these findings illustrate a model-experiment discovery cycle in which quantitative pathway discordance identifies missing biology, revealing a TANK-dependent IKKε checkpoint that restrains MyD88-driven inflammation.