Research ArticleCELLULAR NEUROSCIENCE

Macrophage centripetal migration drives spontaneous healing process after spinal cord injury

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Science Advances  15 May 2019:
Vol. 5, no. 5, eaav5086
DOI: 10.1126/sciadv.aav5086
  • Fig. 1 A time-course RNA-seq analysis reveals prominent IRF8 expression in the spinal cord during the recovery phase after SCI.

    (A) A heat map showing gene expression changes in the injured cord at 4, 7, and 14 dpi. Data from the samples of injured cords were normalized to those of uninjured cords. (B) GO term analysis of overexpressed (>10-fold change) genes in the RNA-seq analysis of the injured cord (7 dpi) compared to those of the uninjured cord. Lists show the top nine GO terms obtained ranked by P value (Fisher’s exact test with Benjamini-Hochberg correction). (C) Volcano plot of the gene expression differences between the injured cord (7 dpi) and uninjured cord. Red or blue dots indicate significantly up-regulated or down-regulated genes, respectively. (D) Wiggle plots showing the coverage of each exon for IRF8 before injury and at 4, 7, and 14 dpi. Ref-seq, reference sequencing. (E) Ratio of the fragments per kilobase of exon per million mapped sequence reads (FPKM) value of IRF8 to the mean FPKM value of all expressed genes at each time point after SCI. (F) Significantly increased mRNA expression of IRF8 analyzed with qPCR in spinal cords before and after injury (n = 5 per each time point). The data shown in (A) to (E) are representative of four samples per time point. *P < 0.05 and **P < 0.001, one-way analysis of variance (ANOVA) with Dunnett’s test for multiple comparisons against the uninjured control group (F). The data are presented as the means ± SEM.

  • Fig. 2 IRF8 is expressed in CD68+ macrophages after SCI.

    (A) Representative images of immunofluorescent staining of spinal cord with CD68 (green) and IRF8 (red) in WT mice at 4 and 7 dpi. The asterisks indicate the lesion epicenter. (B) CD68 (green) and IRF8 (red) double-positive macrophages in the perilesional areas. The nuclei were counterstained with Hoechst 33258 dye (blue). Selective localization of IRF8 in the nuclei of macrophages was observed at 7 dpi (bottom). All IRF8-expressing cells were CD68+. (C) Fluorescence ratio (Fl. ratio) of nuclear IRF8 to cytosolic IRF8 (n = 30 cells per group). (D to H) Double immunostaining of IRF8 with PMN, CD3, GFAP, NeuN, and GST-π. (I) The IRF8 expression examined by reverse transcription PCR (RT-PCR) in selectively isolated macrophages, neutrophils, reactive astrocytes, and neurons. Among these cells, the IRF8 expression was observed only in macrophages. The data shown in (I) are representative of three samples from three mice/each cell fraction. Images shown in (A), (B), and (D) to (H) are representative of eight sections per four mice. The images were obtained from two independent experiments. Scale bars, 500 μm (A), 20 μm (E and G), and 10 μm (B, D, F, and H). **P < 0.005, Wilcoxon rank-sum test. The data are presented as means ± SEM. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

  • Fig. 3 IRF8 is required for macrophage migration toward the epicenter in the injured spinal cord during the recovery phase.

    (A) CD45-gated cells in the injured cord were divided into CD11b+/CD45high/Ly6G infiltrating macrophages. The cell numbers of these macrophages in the injured cord of WT/IRF8−/− mice were quantitatively counted using the FACS software program. FITC, fluorescein isothiocyanate; PE, phycoerythrin; SSC, side scatter; APC, allophycocyanin. (B) The cell numbers of macrophages in the injured cord of IRF8−/− mice were comparable to those of WT mice after SCI (n = 6 per group). Although IRF8−/− mice generate reduced numbers of monocytes/macrophages (66), these results indicate that macrophage infiltration into the injured spinal cord does not require IRF8. n.s., not significant. (C) Change in the distribution of CD68+ macrophages over time. The location of macrophages shifted toward the lesion epicenter from 4 to 14 dpi after injury in WT mice, whereas the macrophages of IRF8−/− mice remained widely scattered in the injured cord. The fine lines indicate the outlines of the spinal cords. Asterisk indicates the epicenter of the lesion. (D) Distribution of macrophages in WT or IRF8−/− mice at 42 dpi. The fine lines indicate the outlines of the spinal cords. Asterisk indicates the epicenter of the lesion. (E) Comparison of the craniocaudal distribution of macrophages in injured cords between WT and IRF8−/− mice (n = 8 WT, 7 IRF8−/− mice per group at each time point, respectively). (F) EGFP+-IRF8+/+ (left) or EGFP+-IRF8−/− (right) monocytes isolated from the peripheral blood of EGFP+-IRF8+/+ or EGFP+-IRF8−/− mice as EGFPposi/CD11bposi/Ly6Gnega/CD115posi monocytic population (fig. S3), respectively, were injected into 2 mm rostral to the epicenter of the injured spinal cord of WT mice once immediately after SCI. Representative images of the immunofluorescent staining of spinal cord injected with these monocytes after SCI. Tp; transplantation of EGFP+ monocytes. The fine broken lines indicate the outlines of the spinal cords. The coarse broken lines indicate the area of the EGFP/CD68+ host macrophages. The asterisks indicate the epicenters of injured spinal cord. The arrowheads indicate the injection point of the EGFP+ macrophages. Inset: High-magnification image. The nucleus was counterstained with Hoechst 33258 dye (blue). Scale bar, 20 μm. The images shown are representative of six slides from three mice per time point per group. (G) Distances of EGFP+-IRF8+/+ or EGFP+-IRF8−/− macrophages from the epicenter at each time point (n = 6 sections/three mice per each time point per group). (H) Schematic representation of intravenous (I.V.) injection of EGFP+-IRF8+/+ or EGFP+-IRF8−/− monocytes after SCI. EGFP+-IRF8+/+ or EGFP+-IRF8−/− monocytes were daily injected intravenously from 1 to 3, 4 to 6, or 7 to 13 dpi. (I) Quantitative analysis of the infiltrating EGFP+ macrophages in the injured cord of mice intravenously injected with EGFP+ macrophages from 1 to 3, 4 to 6, or 7 to 13 dpi (n = 4 sections/four mice per group) (see also fig. S4). The images shown in (C) and (D) are representative of eight or seven slides from eight WT/seven IRF8−/− mice per each time point. Scale bars, 500 μm (C, D, and F). *P < 0.05 and **P < 0.005, Wilcoxon rank-sum test (B), two-way repeated-measures ANOVA with the Tukey-Kramer post hoc test (E and I), and ANOVA with the Tukey-Kramer post hoc test (G). The data are presented as means ± SEM (B, E, G, and I).

  • Fig. 4 Widely scattered macrophages in IRF8−/−mice caused the widespread lesion and the poor functional recovery after SCI.

    (A) 5HT (white) and CD68 (green) staining of the injured cords of WT and IRF8−/− mice at 42 dpi (representative images of seven sections from seven mice per group). (B) Quantification of the 5HT+ area per 4.0 × 105 μm2 at 42 dpi (n = 7 sections/seven mice per group). (C) LFB staining showed a greater demyelinated lesion (arrows) area in IRF8−/− mice than in WT mice at 42 dpi. The asterisk indicates the lesion epicenter. (D) Immunohistochemical staining showed CD68+ macrophages located in the LFB-negative demyelinated area of the serial section shown in (C). The asterisk indicates the lesion epicenter. (E) Quantification of the area of LFB+ myelin at 42 dpi (n = 6 per group). (F) A linear regression analysis showing the negative correlation between the CD68+ and LFB+ areas at 42 dpi (β = −2.49; 95% confidence interval, −3.19 to −1.79). (G) Time course of the functional recovery of the BMS score after SCI (n = 8 per group). (H) A footprint analysis performed at 42 dpi showed that IRF8−/− mice were unable to consistently step. (I) Quantification of the footprint analysis after SCI showing significantly worse motor functions in IRF8−/− mice than in WT mice at 42 dpi (n = 8 per group). (J) Scores for the grip walk test in each mouse at 42 dpi. The images shown in (C) and (D) are representative of six slides from six mice per each group. Scale bars, 50 μm (A) and 500 μm (C and D). *P < 0.05 and **P < 0.005, Wilcoxon rank-sum test (B, E, I, and J) and two-way repeated-measures ANOVA with the Tukey-Kramer post hoc test (G). The data are presented as means ± SEM (B, E, G, and I).

  • Fig. 5 Macrophages migrate to high concentrations of complement component C5a at the lesion epicenter via purinergic receptors.

    (A) Representative images of the immunohistochemical analysis of the spatiotemporal distribution of CD68 (green) and C5a (white) in the injured cord after SCI. The C5a+ area was located medially in the CD68+ area. (B) The C5a expression patterns were dependent on the distance from the lesion epicenters. The C5a expression gradient was observed at 4 and 7 dpi, and the expression of C5a peaked at the lesion epicenter at 7 dpi (n = 8 samples from eight mice/each group/each time point). (C) Representative images of the immunohistochemical analysis of the injured spinal cord obtained from WT mice daily injected with C5a rostral and caudal to the lesion epicenter from 4 to 7 dpi. CD68+ macrophages were attracted to the C5a injection sites. The arrowheads indicate the injection sites of PBS or C5a. (D) A comparison of the craniocaudal distribution of macrophages between the C5a- and PBS-injected spinal cord at 7 dpi (n = 8 samples from eight mice per group). (E) Time-lapse phase-contrast microscopy image sequences of macrophages in the C5a gradient. WT macrophages migrated toward higher concentrations of C5a, whereas IRF8−/− macrophages did not migrate when exposed to a C5a chemotactic gradient. (F) Migration plots of WT (black dots and lines) and IRF8−/− (red dots and lines) macrophages in a gradient of C5a. The starting point of each track was normalized to the position x = 0 and y = 0. The positive values on the y axis represent movement in the direction of the source of C5a (n = 18 per group). (G) Mean velocities of WT and IRF8−/− macrophages treated with C5a (n = 18 per group). (H) Mean chemotaxis indices of WT and IRF8−/− macrophages treated with C5a (n = 18 per group). (I) mRNA expression in the isolated WT and IRF8−/− macrophages (n = 6 samples per group). (J) Representative images of the immunohistochemical analysis of the CD68 macrophages after blockade of purinergic signaling in the injured cord. Inhibitors of purinergic receptors or PBS were injected every other day from 4 dpi. (K) Quantitative analysis of craniocaudal range of CD68+ macrophages with PBS injection (control), triple block, or cocktail block (n = 10 samples from 10 mice per group). The images shown in (A) and (C) are representative of eight slides from eight mice per group or time point. Scale bars, 500 μm. *P < 0.05 and **P < 0.005, Wilcoxon rank-sum test (D, G, H, and I) and two-way repeated-measures ANOVA with the Tukey-Kramer post hoc test (K). The data are presented as means ± SEM.

  • Fig. 6 Activating IRF8 promotes macrophage migration during the recovery phase after SCI.

    (A) Macrophage transplantation protocol. Using a cell sorter, IRF8-activated and nonactivated macrophages were isolated from injured cords at 4 and 7 dpi of EGFP+ bone marrow chimeric mice, respectively. These cells were transplanted into the spinal cord of EGFP WT mice immediately after SCI. (B) Representative images of the immunohistochemical analysis of injured spinal cords transplanted with IRF8-activated or nonactivated macrophages. The arrowheads indicate the injection sites of macrophages. (C) Craniocaudal distribution of the transplanted IRF8-activated macrophages compared with the transplanted nonactivated macrophages in the injected spinal cord at 7 dpi (n = 8 sections/four mice per group). (D) Immunocytochemical analysis of the IRF8 translocation into the nuclei of macrophages differentiated from isolated CD11b+/CD45high/Ly6G peripheral blood–derived monocytes 3 hours after IFN-γ and LPS combination stimulation, stained with CD68 (green), IRF8 (red), and Hoechst (blue). (E) Fluorescence ratio of nuclear IRF8 to cytosolic IRF8 (n = 30 cells per group). (F) mRNA expression of purinergic receptors in macrophages 3 hours after combination stimulation (n = 8 dishes per group). (G) Schedule of in vivo combination injection after SCI. PBS or combination was injected into the spinal cord after SCI. (H) Representative images of the immunohistochemical analysis of the injured cord receiving the combination injection. CD68+ macrophages with combination injection rapidly migrated toward the lesion epicenter at 4 dpi. Increased nuclear translocation of IRF8 of macrophages was shown in the injured cord with combination injection. Scale bars, 500 μm (left) and 10 μm (right). (I) Ratio of nuclear IRF8 to cytosolic IRF8 of macrophages in the injured cord with PBS or combination injection at 4 dpi (n = 30 cells from eight slides per eight mice per group). (J) Time course of macrophage migration in the injured cord with the combination injection. (K) Comparison of the craniocaudal distribution of WT/IRF8−/− macrophages with combination injection and those with PBS injection (n = 8 per group per time point). (L) Representative images of the immunofluorescent staining of spinal cord with PBS/combination injection at 14 dpi. (M) Quantification of the 5HT+ area of spinal cord with PBS/combination injection at 14 dpi (eight sections/eight mice per group). (N) BMS score of WT mice with PBS or combination injection (n = 8 mice per group). (O) Footprint analysis of WT/IRF8−/− mice with combination injection compared to those with PBS injection at 14 dpi. (P) Stride length of WT/IRF8−/− mice with PBS and combination injection at 14 dpi (n = 14 mice per group). (Q) Scores of the grip walk test in each mouse at 14 dpi (n = 14 per group). Scale bars, 500 μm (B and J), 10 μm (D), and 100 μm (L). The images shown in (H), (J), and (L) are representative of eight slides from eight mice/each group per time point. *P < 0.05 and **P < 0.005, Wilcoxon rank-sum test (E, F, I, and M), ANOVA with the Tukey-Kramer post hoc test (C, K, P, and Q) and two-way repeated-measures ANOVA with the Tukey-Kramer post hoc test (N). The data are presented as means ± SEM. (C, F, K, M, N, P, and Q).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/5/eaav5086/DC1

    Fig. S1. Comparable numbers of microglia, Ly6Chigh and Ly6Clow populations, and gene expression levels of M1/M2 markers in macrophages between WT and IRF8−/− mice after SCI.

    Fig. S2. The microglial IRF8–deficient does not affect CD68+ macrophage migration and functional recovery after SCI.

    Fig. S3. Gating for EGFP+ peripheral blood monocytes.

    Fig. S4. Macrophages infiltrated into the injured spinal cord by 3 dpi.

    Fig. S5. Widespread IRF8−/− macrophage with few 5HT+ axons, leading to less demyelination after SCI.

    Fig. S6. KEGG pathway analysis detected six cytokine-related pathways in analysis with RNA-seq data of 4, 7, and 14 dpi.

    Fig. S7. IRF8 has no significant influence on expression levels of genes present in the cytokine-cytokine receptor interaction of KEGG pathway after SCI.

    Fig. S8. IRF8 has no significant influence on expression levels of genes present in the nuclear factor κB signaling pathway of KEGG pathway after SCI.

    Fig. S9. IRF8 has no significant influence on expression levels of genes present in the chemokine signaling pathway of KEGG pathway after SCI.

    Fig. S10. IRF8 has no significant influence on expression levels of genes present in the TLR signaling pathway of KEGG pathway after SCI.

    Fig. S11. IRF8 has no significant influence on expression levels of genes present in the transforming growth factor–β signaling pathway of KEGG pathway after SCI.

    Fig. S12. IRF8 has no significant influence on expression levels of genes present in the TNF signaling pathway of KEGG pathway after SCI.

    Fig. S13. Gene expression levels of cytokines in WT and IRF8−/− macrophages.

    Fig. S14. Gene expression levels of cytokines in WT and IRF8−/− macrophages at 7 dpi.

    Fig. S15. Gene expression levels of cytokines in WT and IRF8−/− macrophages at 14 dpi.

    Fig. S16. The failure of centripetal migration of macrophages led to a wide range of neuronal loss after SCI.

    Fig. S17. IRF8 did not significantly influence expression of C5a receptors of macrophages after SCI.

    Fig. S18. EGFP+ macrophages at 1 hour after transplantation.

    Fig. S19. Promoting IRF8 had no harmful effect on the systemic inflammatory response after SCI.

    Fig. S20. TLR4 signaling of macrophages was activated after SCI.

    Fig. S21. Sources of C5a in injured spinal cord.

    Table S1. Primers used for qPCR.

    Movie S1. Chemotaxis assay of WT macrophages.

    Movie S2. Chemotaxis assay of IRF8−/− macrophages.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Comparable numbers of microglia, Ly6Chigh and Ly6Clow populations, and gene expression levels of M1/M2 markers in macrophages between WT and IRF8−/− mice after SCI.
    • Fig. S2. The microglial IRF8–deficient does not affect CD68+ macrophage migration and functional recovery after SCI.
    • Fig. S3. Gating for EGFP+ peripheral blood monocytes.
    • Fig. S4. Macrophages infiltrated into the injured spinal cord by 3 dpi.
    • Fig. S5. Widespread IRF8−/− macrophage with few 5HT+ axons, leading to less demyelination after SCI.
    • Fig. S6. KEGG pathway analysis detected six cytokine-related pathways in analysis with RNA-seq data of 4, 7, and 14 dpi.
    • Fig. S7. IRF8 has no significant influence on expression levels of genes present in the cytokine-cytokine receptor interaction of KEGG pathway after SCI.
    • Fig. S8. IRF8 has no significant influence on expression levels of genes present in the nuclear factor κB signaling pathway of KEGG pathway after SCI.
    • Fig. S9. IRF8 has no significant influence on expression levels of genes present in the chemokine signaling pathway of KEGG pathway after SCI.
    • Fig. S10. IRF8 has no significant influence on expression levels of genes present in the TLR signaling pathway of KEGG pathway after SCI.
    • Fig. S11. IRF8 has no significant influence on expression levels of genes present in the transforming growth factor–β signaling pathway of KEGG pathway after SCI.
    • Fig. S12. IRF8 has no significant influence on expression levels of genes present in the TNF signaling pathway of KEGG pathway after SCI.
    • Fig. S13. Gene expression levels of cytokines in WT and IRF8−/− macrophages.
    • Fig. S14. Gene expression levels of cytokines in WT and IRF8−/− macrophages at 7 dpi.
    • Fig. S15. Gene expression levels of cytokines in WT and IRF8−/− macrophages at 14 dpi.
    • Fig. S16. The failure of centripetal migration of macrophages led to a wide range of neuronal loss after SCI.
    • Fig. S17. IRF8 did not significantly influence expression of C5a receptors of macrophages after SCI.
    • Fig. S18. EGFP+ macrophages at 1 hour after transplantation.
    • Fig. S19. Promoting IRF8 had no harmful effect on the systemic inflammatory response after SCI.
    • Fig. S20. TLR4 signaling of macrophages was activated after SCI.
    • Fig. S21. Sources of C5a in injured spinal cord.
    • Table S1. Primers used for qPCR.

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). Chemotaxis assay of WT macrophages.
    • Movie S2 (.mp4 format). Chemotaxis assay of IRF8−/− macrophages.

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