Signal regulatory protein α dynamically mediates macrophage polarization facilitated alleviation of ischemic diseases

Figures

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  Fig. 1. LPS and IL-4 promoted angiogenesis and macrophage polarization in a mouse HLI model. After left artery ligation, the mice received PBS, LPS and IL-4 intramuscular injection on day 0, 3 and 7 post-surgery (n = 10). A–C, Laser speckle data showing the relative level of blood perfusion in the hind paws on the indicated days (scale bar: 1000 µm, A and B). Quantitative analyses of the laser speckle images showing the left/right ratio of plantar blood perfusion (D7 and D21, C). D The percentages of dead cells in gastrocnemius muscles from ligated hindlimb on D3, D7, D14, and D21 post-surgery (Tunel Assay, scale bar: 100 µm). E Macrophage were isolated from adductor muscles and the surface markers, CD86 (pro-inflammatory polarization marker), and CD206 (anti-inflammatory polarization marker) were checked using flow cytometry. Total 5000 cells were gated and analyzed. F The phagocytosis of pHrodo Red-labeled hypoxia or nutrition deprivation induced apoptotic mCMVEC by the macrophages isolated from adductor muscles of the mice that received various treatment. Total 5000 cells were analyzed. G, H The cytokines (F) and growth factors (G) secreted from the macrophages that were collected…

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  Fig. 2. LPS and IL-4 promoted angiogenesis in mouse hindlimb ischemia in a time-dependent manner. After left artery ligation, the mice received PBS, LPS, and IL-4 intramuscularly injections at various times. A, B Laser speckle data showing the relative level of blood perfusion in the hind paws of mice that received various treatments on the indicated days (scale bar: 1000 µm). C The percentages of dead cells in gastrocnemius muscles from the mice on D3, D7, D14, and D21 post-surgery, scale bar: 100 µm. D The level of Sirpα in the macrophages collected from adductor muscles of the mice (n = 3). E The phagocytosis of pHrodo Red-labeled hypoxia or nutrition deprivation induced apoptotic mCMVECs by the macrophages from adductor muscles of the mice. Total 5000 cells were gated and analyzed. F, G IL-1α, IL-6, TGF-β (F), and growth factors (G) secreted from the macrophages that were collected from adductor muscles of the mice on the indicated days, were measured using ELISA. PBS, LPS D0 & D4 or IL-4 D0 & D4: the mice received PBS, LPS or IL-4 intramuscular injection on day 0 and day 4 post-surgery. LPS D8 & D12 or IL-4 D8 & D12: the mice received LPS or IL-4 intramuscular injection on day 8 and…

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  Fig. 3. Restored or knockdown Sirpα in the macrophage that received LPS or IL-4, respectively, caused function disorder of the macrophage. Macrophages were isolated from the adductor muscles of wild-type mice. Sirpα was increased or decreased in macrophages that pre-treated with 30 ng/ml LPS or IL-4 for 48 h by Lentivirus (LPS + Sirpα: LPS treatment with Sirpα overexpression or IL-4-Sirpα: IL-4 treatment with Sirpα downregulation), respectively. Lentivirus control was used as the control (LPS + Cont: LPS treatment with Lentivirus control and IL-4 + Cont: IL-4 treatment with Lentivirus control). A, B The level of Sirpα (n = 3) in the macrophages that received LPS (A) or IL-4 (B). C The level of Sirpα (n = 3) in the pre-treated macrophages that were incubated with Lentivirus to manipulate Sirpα expression. D, E The phagocytosisof pHrodo Red-labeled apoptotic mCMVECs by the macrophages were measured by flow cytometry (D, total 5000 cells were analyzed). Quantification of the phagocytosis percentages (E). F, G The cytokines (F) and growth factors (G) secreted from the macrophages that received LPS, LPS + Cont or LPS + Sirpα, were measured using ELISA. H, I The secreted cytokines (H) and…

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  Fig. 4. Sirpα contributed the pro-angiogenic effect of LPS or IL-4 in vivo. A After left femoral artery ligation, PBS, LPS or IL-4 was intramuscularly injected into Sirpα knockout mice (Sirpα−/−) on indicated days post-surgery, WT mice with PBS injection were used as a control (n = 6). Laser speckle data showing the relative level of blood perfusion in the hind paws of mice that received various treatments on the indicated days (scale bar: 1000 µm). B The mice were euthanized 21 days post-surgery. The sections of the gastrocnemius muscle from the ligated side were subjected to immunohistochemistry analysis for CD31 and counterstained with Hoechst 33342 (scale bar: 100 µm, n = 6). Quantification of the CD31+ area. The CD31+ area on the slide from the mouse administered with PBS was set to 1. C Macrophages isolated from WT mice were pre-treated with IL-4 for 48 h and then were injected into ischemic Sirpα−/− mice, ischemic WT mice or Sirpα−/− mice that received IL-4 injection were used as controls (n = 5). Laser speckle data showing the relative level of blood perfusion in the hind paws of mice that received various treatments on the indicated days (scale bar: 1000 µm). D The sections of…

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  Fig. 5. Stat3 or Stat6 mediated to the LPS or IL-4 induced changing of Sirpα. Macrophages from the adductor muscles of wild-type mice were treated with 30 ng/ml LPS or IL-4 at various times. Lentivirus was used to knock down Stat3 (siStat3) or Stat6 (siStat6) in the macrophage. A, B The level of Stat3, phosphorylated Stat3 (pStat3), Stat6, and phosphorylated Stat6 (pStat6) in the macrophages that received LPS for various time (A), and the quantification (B, n = 3). C, D The level of Stat3, pStat3, Stat6, pStat6 in the macrophages that received IL-4 for various times (C), and the quantification (D, n = 3). E and F The levels of miR17 ~ 92 cluster in the macrophages that received LPS (E) or IL-4 (F), were determined by RT-qPCR (n = 4). G Stat3 and Sirpα levels in the LPS pre-treated Stat3 knockdown macrophages were examined. GAPDH was used as a control (n = 3). H Stat6 and Sirpα levels in the IL-4 pre-treated Stat6 knockdown macrophages were examined. GAPDH was used as a control (n = 3). Data is analyzed using one-way ANOVA followed by Tukey multiple range test and expressed as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. ns, non-significant

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  Fig. 6. Macrophage from Sirpα knockout (Sirpα−/−) mice promoted angiogenesis in ischemic WT mice at early stage. A The level of CD47 in the adductor muscles of the mouse that received surgery or sham. GAPDH was used as a control. B The phagocytosis of pHrodo Red-labeled apoptotic mCMVECs (hypoxia or nutrition deprivation induced) by the macrophages from the adductor muscles of the mice that received surgery or sham, were tested by flow cytometry. Total 5000 cells were analyzed. C The phagocytosis of pHrodo Red-labeled apoptotic mCMVECs by the macrophages from the adductor muscles of the Sirp−/− mice or WT mice (Cont). D, E The macrophages collected from the adductor muscles of Sirpα−/− mice were transplanted to the ischemic WT mice on day 0 and day 4 post-surgery (early stage), and PBS was used as a control. Laser speckle data showing the relative level of blood perfusion in the hind paws on the indicated days (D, scale bar: 1000 µm). Quantitative analyses of the images showing the left/right ratio of plantar blood perfusion (E). F The mice were euthanized 21 days post-surgery. The sections of the gastrocnemius muscle from the ligated side were subjected to immunohistochemistry analysis…

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  Fig. 7. Sirpα promoted the stabilization and relocation of HIF1α. The macrophages collected from adductor muscles of WT mice were pre-treated with 30 ng/ml IL-4 for 48 h to elevate the level of Sirpα and then incubated under hypoxic conditions for 0 to 168 h. A The growth factors secreted by the pre-conditioned macrophages were checked using ELISA. B The level of HIF1α in the macrophages’ nuclei at various times post-hypoxic pre-conditioning. C The level of Sirpα in the macrophages at various times post-hypoxic pre-conditioning. D–F Lentiviruses were used to overexpress Sirpα in 30 ng/ml IL-4 pre-conditioned macrophages. Then macrophages were incubated under hypoxic conditions for 0 to 168 h with or without the presence of CD47. The level of HIF1α in the Sirpα overexpressed macrophages nuclei without the presence of CD47 at various times post-hypoxic pre-conditioning (D). The level of HIF1α in the Sirpα overexpressed macrophages nuclei with CD47 at various times post-hypoxic pre-conditioning, Lentiviruses control-treated macrophages were used as a control (E). The nucleus relocation of HIF1α in the macrophages was checked by immunofluorescence (F, n = 4, 168 h post-hypoxic…

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  Fig. 8. LPS and IL-4 worked in a coordinated way to promote angiogenesis and function recovery in the mouse ischemic heart model. Mouse myocardial infarction (MI) was induced by permanently ligating the left anterior descending (LAD) coronary artery. PBS (PBS, n = 8), LPS at early-stage (LPS, n = 6), IL-4 at late-stage (IL-4, n = 9) or LPS at early-stage plus IL-4 at late-stage (LPS (D0) + IL-4 (D7), n = 7) were tail vein injected, respectively. Four weeks post-surgery, cardiac function was evaluated with echocardiography, and hearts were collected thereafter for morphological examination. A and B Infarct size was evaluated with the fibrotic area of the left ventricle by using Masson’s trichrome staining. The blue color represents fibrosis (scale bar: 1000 µm). C Left ventricular fractional shortening (LVFS) was determined with echocardiography. D The level of Sirpα in the infarcted heart was determined by using immunoblotting (n = 4). E The sections of the hearts were subjected to immunohistochemistry analysis of CD31 and counterstained with DAPI (scale bar: 100 µm). The CD31+ area in the PBS group was set to 1. Data is analyzed using one-way ANOVA followed by Tukey multiple range test…