Meta-analysis links TNF pathway proteins to ischemic stroke risk and recurrenceHigher TNF proteins linked to increased stroke risk in large study

Background Inflammation is an emerging target for stroke prevention, but additional therapeutic candidates are needed. Experimental and observational studies implicate tumor necrosis factor (TNF) in atherosclerotic plaque progression and cardiovascular events. We integrated plasma proteomics from population-based studies and prospective stroke cohorts with single-cell and spatial transcriptomic profiling of human atherosclerotic plaque to investigate TNF signaling in stroke pathogenesis. Methods We assessed associations between 34 TNF-superfamily proteins (Olink Explore) and incident ischemic stroke among 47,529 UK Biobank participants without cardiovascular disease. We performed an individual-participant data (IPD) meta-analysis of four prospective cohorts with ischemic stroke (n=2,180) to examine associations between TNF-? and recurrent vascular events. We characterised plaque-level TNF biology using single-cell RNA sequencing of 259,116 cells (62 donors) and Xenium spatial transcriptomics (12 donors) with human carotid plaques. Results In UK Biobank, higher circulating TNF pathway proteins were independently associated with incident stroke after multivariable adjustment, including TNF, TNFR1, and TNFR2 (Hazard Ratio [HR] per SD increase, 1.14, [95% CI 1.07?1.20], 1.22, [1.14?1.31], and 1.15 [1.09?1.21], respectively. An additional 15 TNF superfamily members were also associated with incident stroke. In the IPD analysis of stroke cohorts, TNF-? was associated with recurrent stroke (risk ratio [RR] 1.50, 95% CI 1.14-1.98; top vs. bottom third of TNF-?) and MACE (RR 1.54, 1.18-2.02) after adjustment for cardiovascular risk factors and secondary prevention medications (537 MACE events, 6793 person-years follow up). In single-cell RNA plaque sequencing, TNF and TNF pathway genes were broadly expressed across immune cell populations. In spatial transcriptomics, TNF detection increased progressively from media to fibrous cap (Odds Ratio 2.32 vs media, 95% CI 1.94?2.78; p<0.001). At the fibrous cap, CD8+ effector T cells demonstrated 4.1-fold enrichment for TNF expression despite comprising only 3% of fibrous cap cells. Conclusions TNF signaling is independently associated with incident ischemic stroke and recurrent MACE after stroke. TNF is enriched in human carotid plaque at the fibrous cap, in macrophages and CD8+ effector T cells. These results support evaluation of TNF-targeted therapies for stroke prevention.