Background and Aims

TNF receptor-associated factors (TRAFs) are intracellular signaling adaptor proteins that potently modulate immune cell function and activity in a variety of inflammatory diseases. We and others have shown that distinct members of this family, namely TRAF1, 2, 5 and 6 regulate vascular inflammation and atherogenesis in preclinical studies. However, due to the limited availability of viable animal models, the exact role of TRAF3, TRAF4 and TRAF7 in atherogenesis remains unknown to date. Here, we present a stepwise multiparametric approach to delineate cellular expression, regulation and implications on cardiovascular outcomes of TRAFs in murine and human atherosclerosis by animal experimentation, translational bioinformatical integration of single cell RNA-sequencing (scRNAseq) and complementary clinical studies.

Approach and Results

To induce atherosclerotic lesion formation, LDLR-/- mice were either fed HCD or chow for 20 weeks and expression of TRAF1-7 was determined in whole tissue lysates of the aorta. Notably aortic TRAF3 and TRAF4 expression was 2-fold higher in atherosclerotic mice compared to controls, while TRAF7 expression was significantly lower. In sorted leukocytes from HCD-fed LDLR-/- mice TRAF3 and 4 expression was predominantly increased in B cells and T cells respectively. We then performed scRNAseq of leukocytes from human atherosclerotic plaques and conducted a bioinformatical integration of this human data set with published mouse data to analyze and compare single cell TRAF expression in both species. While the percentage of TRAF expressing cells did not differ significantly for TRAF4 and TRAF7 across species, TRAF3 expression was significantly higher in humans.  Potential significance of cellular TRAF3, 4 and 7 expression in human atherosclerosis was assessed in three independent clinical study cohorts. In patients undergoing coronary angiography TRAF4 blood expression was lower in patients with coronary artery disease (CAD) compared to health individuals. In patients with CAD TRAF7 expression was lower in patients with an unstable CAD angiogram than in patients with stable CAD. TRAF3 expression did not differ in patients with or without CAD as assessed by coronary angiography. Contrastingly, expression of TRAF3 in human atherosclerotic plaques but not of TRAF4 and TRAF7 respectively was significantly higher in unstable atherosclerotic plaques compared to stable lesions. Finally, peripheral mononuclear blood cells from patients with or without a diagnosis of CAD were analyzed for TRAF expression by scRNAseq. Average TRAF3 and TRAF4 expression was lower in patients with CAD compared to healthy controls. This effect was driven by TRAF3 on B cells and myeloid cells and TRAF4 on T cells. Notably, myeloid TRAF3 expression was significantly lower in patients that died from a cardiovascular cause during 20 years of follow up compared with survivors from the same study collective, indicating that cellular TRAF expression may predict cardiovascular long-term outcomes.

Conclusion

Here, we present the first cross-species assessment of cellular TRAF expression in murine and human atherosclerosis. We assess the yet unknown roles of TRAF3, 4 and 7 in atherogenesis by utilizing a multiparametric approach to circumvent the lack of viable knockout models. Finally, we provide first evidence that cellular TRAF expression assessed by scRNAseq may add to the clinicians armamentarium for MACE prediction in the future.