Identification and validation of an endotoxin tolerance–based prognostic model with therapeutic insights in sepsis

Background Sepsis outcomes remain difficult to predict because immune trajectories are heterogeneous and dynamically shift from early activation to immunosuppression. Endotoxin tolerance (ET) in circulating monocytes/macrophages is a key mechanism of sepsis-associated immunosuppression but has not been systematically leveraged for prognostication. We sought to develop and clinically validate an ET-related gene (ETG) signature for short-term mortality risk stratification. Method Public whole-blood transcriptomic datasets were intersected with curated ET gene sets to derive ETG candidates. An ensemble machine-learning framework (108 model/feature-selection combinations across 12 algorithms) was used to build and rank prognostic models for 28-day mortality; the final parsimonious signature (10 ETGs, including IL4R, ATM, CX3CR1, FCGR1A) informed a risk score. A two-variable nomogram (age + ETG risk score) was constructed. Internal performance was assessed by bootstrapped calibration, time-dependent ROC/AUC at 7, 14, and 28 days, and Harrell’s C-index. Experimental validation used a prospective ICU cohort (n=50; 13 survivors, 37 non-survivors). PBMCs were profiled by RT-qPCR and Western blot; CD14 + monocytes were analyzed by flow cytometry for FCGR1A/CD64, CX3CR1, and PD-1. We further explored regulatory context (ceRNA network) and druggability (DGIdb query and molecular docking to CX3CR1, FCGR1A, and TLR5). Results The 10-gene ETG signature stratified mortality risk with acceptable discrimination and calibration. AUCs were 0.76 (95% CI 0.69–0.83), 0.78 (0.72–0.83), and 0.73 (0.67–0.78) at 7, 14, and 28 days, respectively; Harrell’s C-index was 0.782. The age-integrated nomogram showed close agreement between predicted and observed survival across timepoints. Functional enrichment indicated immune-response pathways enriched in the low-risk group. In the clinical cohort, non-survivors exhibited lower mRNA and protein levels of FCGR1A, TLR5, and CX3CR1 in PBMCs; flow cytometry revealed reduced proportions of FCGR1A + , CX3CR1 + , and PD-1 + CD14 + monocytes (p