Bone marrow deficiency of mRNA decaying protein Tristetraprolin increases inflammation and mitochondrial ROS but reduces hepatic lipoprotein production in LDLR knockout mice

Figures

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  Fig. 1. Bone marrow TTP deficiency did not affect food intake but reduced body weight of BM recipient LDLR−/−mice. (A) Schematic representation of TTP−/− bone marrow transplantation (BMT). Eight week-old female LDLR−/− mice were transplanted with TTP−/− or TTP+/+ bone marrow cells. Four weeks after BMT, mice were fed a Western diet for 12 weeks. During the Western diet period, mouse body weight and food intake were measured weekly. (B) Food intake and (C) Growth curves of the mice. The average daily intake of Western diet per mouse was calculated (N = 9 mice per group). Data were presented as mean ± SEM.

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  Fig. 2. Bone marrow TTP deficiency increased neutrophil granulocytes, platelets in peripheral blood, and inflammatory monocytes in the spleen. At the experimental endpoint, peripheral blood was drawn into heparinized tubes by retro-orbital puncture. Blood samples were run on the VetScan HMT hematology analyzer to measure the following parameters: (A, B) Neutrophil granulocyte numbers and percentages; (C, D) Total platelet numbers and platelet hematocrit. (E, F) Flow cytometry analysis of mouse splenocytes. A single cell suspension was made, and cells were stained with anti-CD19 mAb, anti-CD3 PE mAb, anti-CD4 FITC mAb, anti-CD8a FITC mAb, anti-FOXP3 mAb, anti-ly6C FITC mAb, anti-CD11b PE mAb, anti-ly6G FITC mAb, and anti F4/80 FITC mAb to detect different mouse leukocyte populations. Absolute numbers (A) and percentages (B) of various leukocyte subpopulations in the spleen. All data were presented as the mean ± SEM (N = 6 mice each group). *P < 0.05; **P < 0.01, vs WT BM recipient group.

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  Fig. 3. Bone marrow TTP deficiency increased macrophage response to LPS stimulation, and resulted in multi-organ damage and systemic inflammation. (A–B) Residential peritoneal macrophages were obtained from mice at the experimental endpoint. The cells were treated with LPS (50 ng/ml) for 6 h. Total mRNA was extracted for qRT-PCR to measure the expression of inflammatory cytokine TNFalpha (A) and IL6 (B). Data were expressed as mean ± SEM, (N = 3 mice for each group). **p < 0.01. Representative photomicrographs showing H&E staining in the (C) liver (20x), (D) lung (20x), and (E) spleen (10x) in wild-type recipient mice (right panels) and TTP−/− recipient mice (left panels). Scale bar 100 μm. (F) Serum TNFalpha concentrations were determined by ELISA. Data were analyzed by Student's t-test. Data were presented as the mean ± SEM (N = 6 mice each group). ***p < 0.001.

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  Fig. 4. Microarray analysis revealed that bone marrow TTP deficiency in LDLR−/−mice modulated the expression of inflammatory genes, oxidative stress regulators, and lipid metabolism related genes. Mouse liver RNAs were isolated from LDLR−/− mice transplanted with WT or TTP−/− bone marrow cells and fed Western diet for 12 weeks and subjected to Affymetrix microarray analysis. N = 4/group. (A) Heat maps of 236 genes that are significantly changed by more than 2 folds and corrected P-value cut-off:0.05. See Supplementary Fig. S6 for detailed gene list. (B) Heat maps of 27 hepatic genes involved in inflammation and lipid metabolism. Color-coding indicates increased gene expressions in red and decreased gene expressions in green. The columns and rows in the heat maps represent samples and genes, respectively. (C) The network in the ingenuity pathway analysis (IPA) from top regulated pathway “inflammatory response” were shown. Genes that are significantly upregulated by bone marrow TTP deficiency are boxed. The color intensity indicates the fold change expression of genes (red representing upregulation and green representing downregulation). (D) The network in the IPA from top regulated…

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  Fig. 5. Bone marrow TTP deficiency increased mitochondrial reactive oxygen species (mtROS) generation. (A) Venn diagram showed some of the mitochondrial biology related genes from mouse mitocarta gene list which is significantly changed in BM TTP−/− mice based on the microarray data. The mitocarta gene list was collected from the MIT Broad Institute website which includes all the nucleus and DNA encoded mitochondrial genes. (B) Mitochondrial ROS production. BMDMs were generated from bone marrow cells isolated from femurs and tibias of TTP−/− mice and WT littermates. Then, cells were treated with H2O2 (250 μM) for 4 h, then loaded with 5 μM of MitoSOX Red Mitochondrial Superoxide indicator (Life technologies) for 20 min and flow cytometry analysis was performed to quantify mtROS (n = 3). **P < 0.01; ***P < 0.001, vs. control. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

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  Fig. 6. Bone marrow TTP deficiency reduced serum lipid levels, alleviated hepatic steatosis, and reduced cholesterol excretion. (A) Mouse serum total cholesterol and (B) triglyceride levels were measured after 12 weeks of Western diet. (C) Pooled serum samples from mice at the experimental endpoint were analyzed for the lipoprotein profile using FPLC. Cholesterol content in each fraction was determined (n = 6 mice/group). Data represent the mean ± SEM. (D) Very low density lipoprotein (VLDL) and low density lipoprotein (LDL)/high density lipoprotein (HDL) ratio in BM-TTP−/− mice compared to controls. (E) LDL cholesterol/HDL cholesterol ration in cardiovascular disease (CVD) patient compared to non-CVD individuals. Representative Oil Red-O stained liver sections. Scale bar 100 μm. (F) Quantification of Oil Red-O staining. (G) Fecal cholesterol excretion. *p < 0.05; **p < 0.01; ***p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

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  Fig. 7. Bone marrow TTP deficiency had no effects on atherosclerosis after 12 weeks of Western diet feeding in LDLR−/−mice. (A) The lesion area of the frozen sections were detected by H&E staining and quantified by Image-ProPlus 6.0. The quantitative analysis and representative images are shown. (b) Lipid contents in the aortic lesions were determined by ORO staining. Quantitative analysis and representative images are shown. (C) The areas of macrophages in the aortic root lesions were determined by immunostaining for MOMA-2 (a macrophage marker). Quantitative analysis and representative images are shown. (D) Collagen percentage in the aortic root lesions were determined by trichrome staining. Quantitative analysis and representative images are shown. Data were presented as the mean ± SEM (N = 9 mice per group). Scale bar 100 μm.

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  Fig. 8. Schematic representation of a working model. TTP modulates both inflammation and lipid metabolism. Left: During normal physiological condition, TTP acts as an anti-inflammatory molecule by degrading pro-inflammatory TNFalpha mRNA and potentially other pro-inflammatory cytokine mRNAs. Concurrently, TTP increases VLDL/LDL cholesterol indirectly by degrading negative up-regulator INSIG1 mRNA or indirectly by stabilizing HMG-CoA reductase/SREBF1 mRNAs. Right: under pathological conditions, bone marrow TTP deficiency in LDLR−/− mice increased inflammation by stabilizing TNFalpha mRNA and potential other pro-inflammatory cytokine mRNAs and increasing mtROS production. At the same, TTP deficiency indirectly decreases VLDL/LDL cholesterol by potentially stabilizing the insulin induced gene 1 (INSIG1) mRNA or indirectly by destabilizing the mRNA of HMG‐CoA reductase or SREBF1 mRNAs. Increased inflammation, mtROS, and reduced lipids offsets each other, resulting in unchanged atherosclerosis in BM TTP deficient mice.