Quantitative adaptor heterogeneity sets single-cell thresholds for innate immune activation and attenuation

Macrophages exhibit a range of time-dependent responses to external stimuli, but at the single cell-level how receptor input is converted into heterogeneous inflammatory outputs, and how termination occurs remains unclear. Toll-like receptor (TLR) signaling is initiated through supramolecular protein assemblies. In this work, using macrophages with endogenously tagged myeloid differentiation primary response 88 (MyD88), and in primary mouse macrophages, we identify pre-existing heterogeneity in MyD88 abundance as a hidden single-cell variable that shapes the threshold for productive MyD88 assembly and downstream signaling. Strong agonism converts this basal heterogeneity into rapid growth of large MyD88 assemblies, robust NFκB activation, cytokine production, and induction of negative regulators. In contrast, weak agonism produces delayed and less efficient assembly, low-amplitude cytokine output, and weaker feedback engagement, resulting in persistent low-output signaling. Parallel transcriptomic analysis of Alzheimer's disease (AD) brain tissue, and blood samples across additional acute and chronic inflammatory disease cohorts, reveals a consistent, disproportionate activation-relative-to-feedback signature, suggesting incomplete activation-feedback coupling as a correlative but recurring feature of chronic inflammatory disease.