Abstract 219: Apolipoprotein C3 Induces Inflammasome Activation In Human And Mouse Monocytes Only In De-lipidated Form

Abstract

Individuals with diabetes suffer from an elevated risk of coronary artery disease (CAD) events. Increased serum levels of apolipoprotein C3 (APOC3) predict incident CAD in individuals with type 1 diabetes (T1D). APOC3 is a small lipid-binding protein carried by lipoproteins, including VLDL, in circulation. Silencing of APOC3 in a mouse model of T1D-accelerated atherosclerosis has been demonstrated to prevent diabetes-accelerated atherosclerosis, strongly suggesting that APOC3 is a causal mediator. However, the exact mechanism whereby APOC3 promotes atherosclerosis in the setting of T1D is unclear. Mice with T1D demonstrated elevated levels of glucose, triglycerides, APOC3 (non-diabetic [ND] mice=442.6 μg/ml, diabetic [D] mice=755.9 μg/ml, p<0.01, N=21 per group), and IL-18 (ND=124.1 pg/ml, D=256.8 pg/ml, p<0.01, N=18-22 per group) in plasma. The elevated levels of plasma APOC3 and IL-18 in diabetic mice and a recent study demonstrating that de-lipidated APOC3 can stimulate inflammasome activation in human monocytes prompted us to investigate whether lipidated APOC3 has similar inflammasome-activating properties. Our results show that de-lipidated APOC3, but not VLDL (which contains APOC3) significantly stimulates IL-1β release from isolated human monocytes (p<0.01 for vehicle vs APOC3, N=3 per group) and mouse monocytes (p<0.01 for vehicle vs APOC3, N=4-7 per group). Mouse monocytes stimulated with de-lipidated APOC3 also had elevated IL-18 release (vehicle=21.2 pg/mg, APOC3=170.2 pg/mg, p<0.001), Il1b mRNA (APOC3=86.7-fold over control), and Nlrp3 mRNA (APOC3=13.8-fold over control), indicating activation of the NLRP3 inflammasome pathway. However, analysis of serum samples from subjects with T1D showed that virtually all APOC3 is bound to lipoproteins (primarily VLDL and HDL). To further confirm the effect of lipidated APOC3, we bound APOC3 to lipid-based small unilamellar vesicles to mimic the physiological state of APOC3 in plasma. The stimulatory effect of APOC3, measured by IL-1β release from mouse monocytes, was lost. Together, our findings suggest that although de-lipidated APOC3 has the ability to induce inflammasome activation in monocytes, virtually no APOC3 circulates in a lipid-free form in subjects with T1D.