Abstract 584: Allograft Inflammatory Factor-1 is Required for NfκB Pathway Activity in Macrophages and Atherosclerosis

Abstract

Introduction: In preliminary studies, we found that Allograft inflammatory factor-1 (AIF1) supports MΦ migration, phagocytosis, survival and pro-inflammatory cytokine secretion. Moreover, AIF1 limits necrotic core formation in atherosclerotic lesions in vivo . Nuclear Factor-κB (NFκB)-mediated signal transduction has been established at different stages of atherosclerosis. We hypothesize that AIF1-regulated processes in atherosclerosis may be mediated through effects on NFκB signaling. Methods: Bone marrow (BM) derived MΦs were isolated and immortalized from wt and Aif1 -/- mice and stimulated with oxidized-LDL (50 ug/ul; oxLDL). Lysates were immunoblotted for total and phosphorylated (active) p65 NFκB, and for total and phosphorylated forms of the IκBα repressor. Aif1 expression in mouse Raw 264.7 cells was knocked down (KD) using siRNA, and NFκB reporter activity, measured by a luciferase reporter, was assessed after adding LPS+IFN-γ. Immunohistochemical analysis for phospho-p65 NFκB was performed in atherosclerotic lesions (aortic roots) from Apoe -/- (SKO) and Apoe -/- ;Aif1 -/- (DKO) mice maintained on high fat diet for 16 weeks. Results: In AIF1-deficient BMMΦs stimulated with oxLDL, we found no differences in the levels of total p65 NFκB and IκBα, but interestingly, phospho-p65 NFκB levels were significantly reduced and phospho-IκBα levels increased compared to wt cells (P<0.05). AIF1 KD using siRNA significantly reduced NFκB activity compared to scrambled control (scrambled control vs. AIF1 KD; 40% vs. 22% luciferase activity, P<0.05), and this impairment was rescued with the addition of AIF1 cDNA. In vivo , NFκB phospho-p65 staining showed that in comparison to SKO samples, DKO aortic roots had decreased phospho-p65 NFκB (SKO vs. DKO; 7.0 vs. 4.0 +nuclei/aortic root, P<0.05). Conclusions: AIF1 is required for NFκB activation in MΦs and moreover, AIF1 enhances NFκB activity in atherosclerotic lesions in vivo . Because NFkB has been closely linked to both cytokine expression and cell survival signaling, these results point to a critical role for AIF1 in pro-inflammatory MΦ functions. Future studies involve identifying the precise steps of the pathway controlled by AIF1, and the mechanisms by which AIF1 affects NFκB signaling.