A host lipase prevents lipopolysaccharide-induced foam cell formation

Figures

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  Figure 1. LPS-exposed Aoah−/− peritoneal macrophages accumulate more cholesterol Aoah+/+, Aoah−/−, and Aoah−/−Tlr4−/− mice were fed a regular diet (RD) or the HFHC diet. On day 0 they, were injected i.p. with either PBS or 10 μg LPS. On day 14, their peritoneal macrophages were studied. (A) Macrophages were allowed to adhere to cell culture plates and stained with Nile Red. Similar results were observed in 4 experiments, n = 3 in each experiment. Scale bar represents 100 μm. (B) Peritoneal cells were stained with anti-F4/80 antibody and BODIPY. The BODIPY fluorescence intensity of the F4/80+ macrophages was measured using flow cytometry. n = 3 or 4. (C) The amount of cholesterol and triglyceride was measured in adherent macrophages. Data were combined from at least 2 experiments, n ≥ 3/group. Data were presented as mean ± SE. One-way analysis of variance (ANOVA) was used to test the 4 groups (blue stars), and the Mann-Whitney test was used to test difference between groups (black stars). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. See also Figures S1 and S2.

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  Figure 2. LPS-exposed Aoah−/− peritoneal macrophages have dysregulated cholesterol metabolic gene expression (Next paragraph) Mice were injected i.p with 10 μg LPS (A) Twenty-one days later, their peritoneal macrophages were purified and cultured either untreated (Aoah+/+, Aoah−/−) or treated with 1 μg/mL LPS for 2 h ex vivo (Aoah+/+ LPS, Aoah−/− LPS) before mRNA was collected for microarray analysis. The gene expression levels involved in cholesterol metabolism that were significantly different (P < 0.05) in Aoah+/+ vs. Aoah−/− macrophages are shown. n = 3. The gene expression levels of Aoah+/+ macrophages were set to 1, and the fold expression changes were calculated for the other groups. (B–D) Fourteen days after LPS injection, peritoneal macrophages were purified, and qPCR analysis was performed (B). Data were combined from at least 2 experiments. n = 7–10. ApoE, ABCA1 (C), and LDLR (D) proteins in peritoneal macrophages were measured. n = 3. The western blots were repeated with similar results. (E) Peritoneal cells were incubated with BODIPY-labeled LDL and then stained with anti-F4/80 antibody. LDL uptake by macrophages was measured using flow cytometry. n = 5–7. Data were presented…

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  Figure 3. After parenteral LPS exposure, Aoah−/− mice maintain residual inflammatory changes while Aoah+/+ mice recover (A) Fourteen days after they received i.p. LPS injection, Aoah−/− mice had elevated serum chemokines MCP-1 and RANTES, n ≥ 3. (B) Analysis of the same microarray data as in Figure 2A was performed, n = 3. The gene expression levels of Aoah+/+ macrophages were set to equal 1, and fold expression changes were calculated for the other groups. (C and D) Fourteen days after i.p. LPS, Aoah−/− peritoneal macrophages had increased expression of mRNAs for inflammatory genes (C); elevated M1 and decreased M2 marker expression (D). Data were combined from at least 2 experiments, n ≥ 4/group. Data were presented as mean ± SE. One-way ANOVA was used to test the 4 groups (blue stars), and the Mann-Whitney test was used to test difference between groups (black stars). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. See also Figure S4.

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  Figure 4. LPS-exposed Aoah−/− peritoneal macrophages have impaired mitochondrial function Aoah+/+ and Aoah−/− mice were injected i.p. with 10 μg LPS. Fourteen days later, their peritoneal macrophages were explanted. (A) Peritoneal macrophages were treated with medium or 10 ng/mL LPS for 20 h. The medium lactate was measured. Data were combined from 3 experiments, n = 3 in each experiment. (B) Peritoneal macrophages were stained with an adenovirus carrying Cox8a-RFP. Mitochondrial length was measured by using Image-Pro 6.0 Plus software. Approximately, 2000 mitochondrial lengths were measured in total 7 or 8 macrophages from 3 Aoah−/− or Aoah+/+ mice, respectively. Scale bar represents 5 μm. (C) Peritoneal macrophages were allowed to adhere and then fixed and processed for electron microscopy. Note that Aoah−/− macrophages had mitochondrial fragmentation and reduced cristae number. Representative pictures of total 9 macrophages from 3 mice of each genotype are shown. Arrows indicate mitochondria. Scale bar represents 600 nm. (D) Cellular ATP levels were measured. Data were combined from 2 experiments, each with n = 3. (E) On day 14, mouse peritoneal macrophages were stained with MitoTracker…

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  Figure 5. After parenteral exposure to LPS, AOAH prevents foam cell formation in carotid artery lesions (A) Starting on day 0, mice were fed an HFHC diet. On day 7, a silastic collar was placed around the left carotid artery of each mouse. Mice were injected i.p. with PBS or 5 μg LPS on days 14, 28, and 42. They were euthanized on day 56 for analysis. (B) The carotid artery segment proximal to the collar was obtained for histology. Hematoxylin- and eosin-stained cross sections are shown. The lesion areas were quantitated by using Image-Pro 6.0 Plus software. Data were combined from at least 4 experiments, n ≥ 8 mice/group. (C) Cross sections were stained with anti-Mac2 antibody to visualize macrophages. n = 5 or 6 mice/group. (D) After the carotid artery segment proximal to the collar was obtained, the segments were embedded in OCT and frozen at −80°C before they were sectioned for oil red O staining and quantitation. n ≥ 5 mice/group. (E) The intra-collar carotid segments, which also had plaques, were harvested and RNA was extracted for qPCR analysis. Data were combined from at least 3 experiments, n ≥ 9/group. Scale bars represent 100 μM (in B, C, D). Data were presented as mean ± SE.…