Abstract Body (Do not enter title and authors here): Objective: Atherosclerosis is a chronic inflammatory condition. Extracellular nucleotide metabolism by CD39, encoded by the ENTPD1 gene, plays a role in modulating inflammation. Our objective of this study was to elucidate the molecular and inflammatory mechanisms by which CD39 activity modulates atherosclerosis
Approach and Results: Here we identify that the genotype of SNP rs3814159 in the promoter region of the ENTPD1 gene strongly associates with the level of expression of Tregs (p<0.001; GG=Treg CD39low; AA=Treg CD39high). Given the contribution of Tregs to regulating inflammation and atherosclerotic cardiovascular disease, we examined the extent of angiographic atherosclerosis using a 17-segment modified AHA model (Gensini Score) in 85 consecutive patients referred for coronary angiogram. Analysis of atherosclerotic burden by rs3814159 genotype demonstrated a significantly higher burden of disease in those patients with the GG (Treg CD39low) genotype (Fig. 1A: p=0.0373). To study how differences in CD39 activity in Tregs affect atherosclerosis. We generated a cre-lox Treg cell-specific CD39 knockout mouse model with green fluorescent protein (GFP) labeled FoxP3 (FoxP3-Cre GFP+ CD39flox) and crossed this into an Ldlr-/- background. We fed high-fat diet (21% saturated fat, 0.15% cholesterol) to Ldlr-/--FoxP3-Cre+-Cd39wt/wt (Treg Cd39high) and Ldlr-/-- FoxP3-Cre+-Cd39flox/flox (Treg Cd39low) mice for 16 weeks. Examination of the aortic sinus by Oil Red O-stained cross-sections revealed a significantly increased lesion area in Treg Cd39low mice (Fig. 1B: p= 0.0321). Next, using an established model of zymosan-induced peritonitis, macrophage efferocytosis of irradiated splenocytes was examined in vivo. Peritoneal macrophages from Treg Cd39low demonstrated a significant reduction in efferocytosis compared to Treg Cd39high mice (Fig. 1C: p=0.047).
Conclusion: Our data support that a reduced expression of CD39 on Tregs results in increased atherosclerosis burden in humans and mice. The mechanism appears to involve Treg regulation of macrophage efferocytosis.