Research ArticleIMMUNOLOGY

Novel reprogramming of neutrophils modulates inflammation resolution during atherosclerosis

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Science Advances  06 Feb 2019:
Vol. 5, no. 2, eaav2309
DOI: 10.1126/sciadv.aav2309
  • Fig. 1 Subclinical endotoxemia exacerbates atherosclerotic pathogenesis.

    ApoE−/− mice were administrated with PBS or superlow-dose LPS, together with HFD for 4 weeks. (A) Representative images of H&E-stained atherosclerotic lesions and quantification of plaque size demonstrated as the percentage of lesion area within aortic root area. Scale bars, 300 μm. (B) Representative images of Oil Red O–stained atherosclerotic plaques and quantification of lipid deposition within lesion area. Scale bars, 300 μm. (C) Representative images of Picrosirius red–stained atherosclerotic plaques and quantification of collagen content within lesion area. Scale bars, 100 μm. (D) Determination of circulating MMP9, LTB4, and TGFβ levels by enzyme-linked immunosorbent assay (ELISA). Data are representative of two independent experiments, and error bars represent means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001; Student’s t test (n = 5 for each group).

  • Fig. 2 Subclinical endotoxin primes neutrophils into a proinflammatory state in atherosclerotic mice.

    ApoE−/− mice were administrated with PBS or superlow-dose LPS, together with HFD for 4 weeks. The surface phenotypes of Ly6G+ neutrophils in the peripheral blood (A), spleen (B), and BM (C) were analyzed with flow cytometry. Data are representative of two independent experiments, and error bars represent means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, Student’s t test (n = 5 for each group). ns, not significant.

  • Fig. 3 Superlow-dose LPS induces inflammatory polarization of neutrophils in vitro.

    Neutrophils were purified from the BM of wild-type C57BL/6 mice and treated with PBS, superlow-dose LPS (100 pg/ml), and/or oxLDL (10 μg/ml) for 2 days. (A) Levels of MMP9, LTB4, and MPO were determined by ELISA (n = 3 for each group). (B) The surface phenotype of neutrophils was analyzed by flow cytometry (n = 3 for each group). (C) Levels of miR-24 and miR-126 were determined by real-time reverse transcription polymerase chain reaction (RT-PCR) (n = 4 for each group). (D) The expressions of oxCaMKII and 5-LOX were determined by Western blot. (E) Representative histogram and quantification of p-STAT1 level as determined by flow cytometry (n = 3 for each group). (F) Representative histograms and quantification of ATF4 and KLF2 levels as determined by flow cytometry (n = 5 for each group). Data are representative of three independent experiments, and error bars represent means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, (A to C and E) one-way analysis of variance (ANOVA) and (F) Student’s t test.

  • Fig. 4 Neutrophils polarized by superlow-dose LPS aggravate atherosclerosis.

    Neutrophils purified from ApoE−/− mice were treated with PBS or superlow-dose LPS (100 pg/ml) for 24 hours. PBS- or LPS-polarized neutrophils (2 × 106 cells per mouse) were then adoptively transferred by intravenous injection to HFD-fed ApoE−/− mice once a week for 4 weeks. Samples were collected 1 week after the last neutrophil transfer. (A) Representative images of H&E-stained atherosclerotic lesions and quantification of plaque size exhibited as the percentage of lesion area within aortic root area. Scale bars, 300 μm. (B) Representative images of Oil Red O–stained atherosclerotic plaques and quantification of lipid deposition within lesion area. Scale bars, 300 μm. (C) Representative images of Picrosirius red–stained atherosclerotic plaques and quantification of collagen content within lesion area. Scale bars, 100 μm. (D) Representative images and quantification of lesional oxCaMKII levels by confocal microscopy. Scale bars, 100 μm. DAPI, 4′,6-diamidino-2-phenylindole. (E) Determination of circulating MPO, MMP9, and LTB4 levels by ELISA. Data are representative of two independent experiments, and error bars represent means ± SEM. *P < 0.05 and **P < 0.01, Student’s t test (n = 5 to 6 for each group).

  • Fig. 5 4-PBA enhances peroxisome homeostasis in neutrophils.

    Neutrophils were purified from the BM of wild-type C57BL/6 mice and treated with PBS, superlow-dose LPS (100 pg/ml), and/or 4-PBA (1 mM) for 2 days. (A) Representative histogram of ROS level determined by CellROX labeling. (B) Quantification of ROS levels in neutrophils (n = 3 for each group). (C) Representative confocal microscopy images of the neutrophils stained with anti-PMP70 (peroxisomal membrane protein 70) and anti-LAMP1 (lysosomal-associated membrane protein 1) antibodies to demonstrate the localization and fusion of peroxisomes and lysosomes. Scale bars, 5 μm. (D) Western blot data of oxCaMKII and 5-LOX expression. (E) Representative histograms and quantification of p-STAT1 (n = 3 for each group), ATF4, and KLF2 (n = 5 for each group) levels as determined by flow cytometry. (F) Surface phenotype of neutrophils was analyzed by flow cytometry (n = 3 for each group). (G) The levels of MPO, MMP9, and LTB4 were determined by ELISA (n = 3 for each group). Data are representative of three independent experiments, and error bars represent means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, one-way ANOVA.

  • Fig. 6 Neutrophils reprogrammed by 4-PBA alleviate atherosclerosis.

    Neutrophils purified from ApoE−/− mice were treated with PBS or 4-PBA (1 mM) for 24 hours. PBS- or 4-PBA–polarized neutrophils (2 × 106 cells per mouse) were then adoptively transferred by intravenous injection to HFD-fed ApoE−/− mice once a week for 4 weeks. Samples were collected 1 week after the last neutrophil transfer. (A) Representative images of H&E-stained atherosclerotic lesions and quantification of plaque size exhibited as the percentage of lesion area within aortic root area. Scale bars, 300 μm. (B) Representative images of Oil Red O–stained atherosclerotic plaques and quantification of lipid deposition within lesion area. Scale bars, 300 μm. (C) Representative images of Picrosirius red–stained atherosclerotic plaques and quantification of collagen content within lesion area. Scale bars, 100 μm. (D) Determination of circulating MPO, MMP9, LTB4, and TGFβ levels by ELISA. (E) Determination of circulating miR-24 and miR-126 levels by real-time RT-PCR. Data are representative of two independent experiments, and error bars represent means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, Student’s t test (n = 5 to 7 for each group).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/2/eaav2309/DC1

    Fig. S1. Subclinical endotoxin up-regulates MPO level in HFD-fed mice.

    Fig. S2. Subclinical endotoxemia exacerbates atherosclerotic pathogenesis in RD-fed mice.

    Fig. S3. Subclinical endotoxin causes neutrophil expansion in atherosclerotic mice.

    Fig. S4. Subclinical endotoxin primes neutrophils into a proinflammatory state in atherosclerotic mice.

    Fig. S5. Subclinical endotoxin induces oxCAMKII elevation in vivo.

    Fig. S6. Neutrophils maintain viability after in vitro polarization.

    Fig. S7. Transfusion of superlow-dose LPS–polarized neutrophils elevates plasma lipid levels and modulates lesional macrophages.

    Fig. S8. Superlow-dose LPS and oxLDL treatment elevates ROS accumulation in neutrophils.

    Fig. S9. 4-PBA reverses superlow-dose LPS–induced differential regulation of miR-24 and miR-126 in neutrophils.

    Fig. S10. Transfusion of 4-PBA–polarized neutrophils down-regulates plasma lipid levels and reduces lesional macrophage activation.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Subclinical endotoxin up-regulates MPO level in HFD-fed mice.
    • Fig. S2. Subclinical endotoxemia exacerbates atherosclerotic pathogenesis in RD-fed mice.
    • Fig. S3. Subclinical endotoxin causes neutrophil expansion in atherosclerotic mice.
    • Fig. S4. Subclinical endotoxin primes neutrophils into a proinflammatory state in atherosclerotic mice.
    • Fig. S5. Subclinical endotoxin induces oxCAMKII elevation in vivo.
    • Fig. S6. Neutrophils maintain viability after in vitro polarization.
    • Fig. S7. Transfusion of superlow-dose LPS–polarized neutrophils elevates plasma lipid levels and modulates lesional macrophages.
    • Fig. S8. Superlow-dose LPS and oxLDL treatment elevates ROS accumulation in neutrophils.
    • Fig. S9. 4-PBA reverses superlow-dose LPS–induced differential regulation of miR-24 and miR-126 in neutrophils.
    • Fig. S10. Transfusion of 4-PBA–polarized neutrophils down-regulates plasma lipid levels and reduces lesional macrophage activation.

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