Abstract 3039: TOMM40 Knockdown In Macrophages Inhibits Oxidized LDL-induced NLRP3 Activation And Promotes Reverse Cholesterol Transport

Abstract

Atherogenesis has been shown to be promoted by activation of the NLRP3 inflammasome, a cytosolic innate immune sensor activated by a broad range of factors, including oxidized LDL (oxLDL). Our group has shown that hepatocyte knockdown of Translocase of Outer Mitochondrial Membrane 40 ( TOMM40), which encodes a key component of the mitochondrial importer TOM complex, results in increased levels of oxysterols that activate the transcription factors LXRα and β. As LXR agonists have been shown to inhibit NLRP3 activation, we tested whether TOMM40 knockdown reduces NLRP3 activation in THP-1 monocyte-derived macrophages. On stimulating these cells with both the NLRP3 activator Nigericin and oxLDL following TOMM40 knockdown by siRNA, we found that pro-inflammatory cytokine release and NLRP3 activation were significantly inhibited (Two-way ANOVA, p<0.0001, n=6). We further showed that TOMM40 knockdown resulted in significant LXRβ upregulation (unpaired t-test, p=0.007, n=3), and reduced oxLDL-induced upregulation of both NLRP3 and CASP1 , which encodes a protein recruited in NLRP3 activation (One-way ANOVA, p=0.0007, n=3). Moreover, pre-treatment with an LXR antagonist, GSK2033, abrogated TOMM40 knockdown-induced NLRP3 inhibition, supporting our hypothesis that this inhibition is likely LXR dependent. Since LXRs are known to promote cholesterol efflux from macrophages, we also examined the effect of TOMM40 knockdown on expression of the cholesterol transporter genes, ABCA1, ABCG1, and SR-B1 , and found that ABCG1 and SR-B1 were significantly upregulated (unpaired t-test, p=0.02 and p=0.003 respectively, n=3). Taken together, these findings identify mechanisms by which TOMM40 knockdown exerts anti-inflammatory effects and promotes reverse cholesterol transport. While TOMM40 has been studied largely in relation to APOE and neurodegenerative disease due to its genomic location, we identify a novel potential impact of TOMM40 on atherogenesis.