Lipopolysaccharide and imiquimod stimulation of potential immune biomarkers in whole blood predict alcohol use disorder risk based on AUDIT scores

Background Alcohol use disorder (AUD) lacks objective clinical tests; current screening (AUDIT) relies on self‐report and can miss risk. Building on our recent whole‐blood analysis, where immune dysregulation, particularly IL‐1β, predicted AUD risk, we tested whether Toll‐like receptor (TLR) stimulation would further unmask risk‐related immune signatures. Methods Whole blood from 28 young adults (Low‐risk: AUDIT <6; High‐risk: AUDIT ≥6) was stimulated in culture with lipopolysaccharide (LPS, TLR4) or imiquimod (IMQ, TLR7). Fourteen immune mediators were quantified using Luminex multiplex assays. Group and stimulus effects were tested with aligned rank transform (ART) factorial models; principal component analysis (PCA) summarized the multivariate structure. Predictive associations with AUDIT were assessed via linear regression and Random Forest analyses. Results IL‐1β, IL‐3, IL‐6, IL‐7, IL‐8, IL‐18, CCL11, MCP‐1, and MIP‐1β were elevated in the high‐risk group following stimulation. LPS evoked stronger responses than IMQ for IL‐1β, IL‐6, IL‐8, and MIP‐1β, whereas MCP‐1 was higher with IMQ. PCA distinguished high‐ from low‐risk groups, driven by CCL11, MCP‐1, IL‐7, IL‐3, IL‐6, IL‐18, MIP‐1β, and IL‐1β. LPS‐evoked IL‐1β, IL‐3, and CCL11 predicted AUDIT scores (adjusted R 2 = 0.22–0.37). IMQ‐evoked CCL11, IL‐18, MIP‐1β, IL‐1β, and IL‐6 were also significant predictors (adjusted R 2 = 0.16–0.29). After outlier filtering, LPS associations persist, and IMQ‐evoked CCL11 and IL‐18 remain. Random Forests predicted AUDIT with R 2 = 0.33 (LPS) and R 2 = 0.27 (IMQ), with top features IL‐3, IL‐18, IL‐1β, CCL11 (LPS) and IL‐6, IL‐18, CCL11, IL‐1β (IMQ). Conclusions LPS and IMQ stimulations unmasked immune response patterns that separated high‐ from low‐risk individuals, with exaggerated pro‐inflammatory responses in the high‐risk group. IL‐1β, IL‐3, IL‐18, and CCL11 repeatedly predicted AUDIT scores, with IL‐1β and IL‐3 LPS‐dominant and CCL11 and IL‐18 LPS/IMQ stimulus‐shared. Stimulation‐evoked mediator profiling may complement self‐report screening and improve risk stratification in drinkers. Further studies are needed to address the exploratory nature of the results.