Abstract
BACKGROUND: Although inflammatory cytokines are pivotal to the pathogenesis of sepsis, determining their causal roles remains challenging due to confounding biases. We employed Mendelian randomization (MR) to investigate genetically determined cytokine levels in sepsis risk, with translational validation in clinical cohorts and experimental models. METHODS: A multi-omics framework integrated cis-protein quantitative trait loci (cis-pQTL) of plasma cytokines with the UK Biobank sepsis GWAS using inverse-variance weighted MR and Wald ratio methods. Sensitivity analyses, Bayesian co-localization analysis, phenotype scanning, and bidirectional MR ensured robustness. Clinical validation compared peripheral levels of the result found by MR analysis in severe sepsis patients (n = 15) and non-septic ICU controls (n = 11) within 24 hours of diagnosis. The temporal dynamics were further characterized in the cecal ligation and puncture (CLP) rat model, assessing blood and lung protein and mRNA levels of the result found in MR analysis at 24 hours to 120 hours, along with T-cell exhaustion markers. RESULTS: Genetically elevated levels of CCL4 (Chemokine CC motif ligand 4) were associated with critical sepsis risk (OR = 0.70, 95% CI: 0.58-0.84, P = 1.45×10(-4), FDR = 0.017), consistent across sensitivity analyses. Clinically, septic patients exhibited higher peripheral blood levels of CCL4 within 24 hours than controls. In the CLP rat model, peripheral and pulmonary CCL4 protein levels peaked at 24 hours but declined significantly by 120 hours. This decline was accompanied by transcriptomic evidence of T-cell exhaustion, with increased CTLA-4 and decreased IL-2 and IFN-γ. CONCLUSION: The trajectory of CCL4 follows distinct phases in sepsis-its early elevation is associated with hyperinflammation, while its later decline correlates with T-cell exhaustion. Although causal mechanisms require further validation, our findings propose that monitoring CCL4 dynamics may serve as a potential biomarker for immunophenotype stratification, highlighting its relevance for developing time-sensitive therapeutic strategies.