Transcriptomic profiling identifies tumor necrosis factor receptor-associated factor (TRAF)-5 expression in human atherosclerotic plaques as a predictor of future cardiovascular events

Background Atherosclerosis is driven is driven by chronic inflammation within the vessel wall. Tumor necrosis factor receptor-associated factors (TRAFs) are cytoplasmic adapter proteins that modulate immune cell function by integrating and regulating receptor-mediated signaling downstream of TNF receptors, interleukin-1 receptors, and Toll-like receptors. While preclinical experimental data implicate a significant role of TRAFs in atherogenesis, their clinical relevance in human atherosclerosis remains unkown. Purpose To evaluate TRAF1–7 expression in human atherosclerotic plaques and determine associations with plaque phenotype, cellular mechanisms, and future cardiovascular events. Methods TRAF1–7 expression was analyzed in early and advanced carotid plaques by bulk transcriptomics. Prognostic relevance of lesional TRAF expression was tested in 616 carotid endarterectomy specimen from the Athero-Express Biobank by multivariable cox regression adjusted for age, sex, diabetes, diastolic blood pressure, eGFR, and peripheral arterial disease. Major adverse cardiovascular events (MACE) were defined as a composite of nonfatal myocardial infarction, stroke, and cardiovascular death within 3 years after surgery. Cellular sources and potential immune cell mechanisms were explored using single-cell RNA sequencing (scRNA-seq) of 259,493 cells from 79 human plaques. Results Among all TRAFs, only TRAF5 expression was significantly reduced in advanced versus early lesions (A, B). Higher plaque TRAF5 levels were independently associated with a lower risk MACE during a follow-up period of 3-years (HR 0.62, 95%CI 0.40 – 0.98) (C). TRAF5 expression was lowest in fibro-inflammatory and unstable plaques, linking reduced TRAF5 levels to increased plaque vulnerability (D, E). scRNA-seq analysis identified B cells as the major TRAF5-expressing cell population (F, G). TRAF5+ B cells exhibited downregulation of pro-inflammatory pathways including IFNγ-, IL-32-, and SPP1-related signaling, reduced chemokine expression and decreased immunoglobulin transcripts. In contrast, immunoregulatory mediators such as TGF-β2, IL-13 and SIAE were enriched (H). Gene set enrichment and pathway analyses confirmed attenuation of inflammatory activation and B-cell receptor signaling in TRAF5+ B cells (I, J). Conclusion TRAF5 expression identifies a less inflammatory, more stable plaque phenotype and independently predicts reduced cardiovascular risk after carotid endarterectomy. Within atherosclerotic lesions TRAF5 marks a distinct immunoregulatory B-cell state and may serve as a future biomarker and potential target for personalized risk stratification in atherosclerotic cardiovascular disease.