Fig 2: Elevated oxidation status in peritoneal macrophages from ApoE-/-and APOE4 mice. Peritoneal macrophages were isolated from WT, ApoE-/- (EKO), APOE2, APOE3, and APOE4 gene replacement mice (N = 7) to assess redox status by measuring reduced (GSH) and oxidized (GSSG) GSH levels and hydrogen peroxide levels. A, reduced GSH and oxidized GSH (GSSG) levels in WT and EKO macrophages; (B) hydrogen peroxide levels in WT and EKO macrophages; (C) GSH and GSSG levels in APOE gene replacement mice; and (D) hydrogen peroxide levels in APOE gene replacement mice. Statistical significance was evaluated by comparing WT and EKO mice with Student t test. Differences between APOE gene replacement mice were evaluated by one-way ANOVA with Student–Newman–Keuls post hoc test. apoE, apolipoprotein E.

Fig 3: Increased lipid rafts in apoE-deficient and apoE2- and apoE4-expressing macrophages. Peritoneal macrophages isolated from WT, ApoE-/- (EKO), APOE2, APOE3, and APOE4 gene replacement mice were stained for GM1 with cholera toxin to identify lipid rafts. Representative images (scale bar = 50 µm) of data from four experiments are shown. Statistical significance was evaluated by comparing WT and EKO mice with Student's t test. Differences between APOE gene replacement mice were evaluated by one-way ANOVA with the Student–Newman–Keuls post hoc test. apoE, apolipoprotein E.

Fig 4: ApoE deficiency or apoE2 expression in macrophages enhances inflammasome priming and activation.A, macrophages isolated from WT and ApoE-/- (KO) mice (N = 4) were incubated in the absence (-) or presence of 100 ng/ml LPS. Extracts were prepared for Western blot analysis of NLRP3 levels using ß-actin as the control. B, macrophages from WT and KO mice (N = 8) were incubated under basal conditions or in the presence of LPS with or without ATP stimulation. IL1-ß secreted into the media was measured by ELISA. C, lysates prepared from WT and KO macrophages incubated with LPS with or without ATP were used for Western blot analysis with antibodies against caspase-1 (CASP1) to identify pro-caspase1 and its cleavage products as indicated. Similar experiments were performed with human APOE2, APOE3, and APOE4 gene replacement mice, and results obtained include the following: (D) NLRP3 levels (N = 3); (E) IL-1ß secretion under basal conditions (N = 10) or in the presence of LPS with (N = 6) or without (N = 7) ATP; (F) MCP-1 and MIP-1a mRNA levels in LPS-stimulated macrophages; and (G) caspase-1 cleavage analysis in LPS/ATP-stimulated macrophages. Extracts were also prepared from splenocytes isolated from 4 WT and four KO mice (H) or human APOE2, APOE3, and APOE4 gene replacement mice (I) and subjected to Western blot analysis with antibodies against caspase-1 to identify pro-caspase1 and its cleavage product as indicated. All data were evaluated by one-way ANOVA with Student–Newman–Keuls post hoc test for significant differences between groups as indicated. apoE, apolipoprotein E; IL, interleukin; LPS, lipopolysaccharide; NLRP3, NOD-, LRR-, and pyrin domain–containing protein 3.

Fig 5: Enhanced myelopoiesis in mice with apoE2-expressing myeloid cells. Blood was obtained from WT and ApoE-/- (EKO), APOE2, APOE3, and APOE4 gene replacement mice (N = 8–10 in each group) for analysis by automatic white blood cell counter to determine the number of (A) total white blood cells in WT and KO mice; (B) neutrophils in WT and KO mice; (C) monocytes in WT and KO mice; (D) lymphocytes in WT and KO mice; (E) total white blood cells in APOE2, APOE3, and APOE4 gene replacement mice; (F) neutrophils in the gene replacement mice; (G) monocytes in the gene replacement mice; and (H) lymphocytes in the gene replacement mice. I, bone marrow cells isolated from the gene replacement mice (N = 6 for apoE2 and apoE3 and N = 4 for apoE4 mice) were cultured for 7 days in the presence of cytokines to promote granulocyte/monocyte colony formation to determine GM-CFU. Statistical significance was evaluated by comparing WT and EKO mice with Student's t test. Comparisons between APOE gene replacement mice were evaluated by one-way ANOVA with Student–Newman–Keuls post hoc test. apoE, apolipoprotein E;