Research ArticleCELLULAR NEUROSCIENCE

Microglia response following acute demyelination is heterogeneous and limits infiltrating macrophage dispersion

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Science Advances  15 Jan 2020:
Vol. 6, no. 3, eaay6324
DOI: 10.1126/sciadv.aay6324
  • Fig. 1 Fate mapping as a tool to specifically label microglia/CAMs.

    (A and B) Representative immunohistochemical images of the uninjured spinal cord demonstrated very high recombination efficiency at 4 weeks after tamoxifen (tdTom reporter, red; IBA1, green) (A). This observation was also reflected in quantification (B). (C and D) Flow quantification of reporter expression in the blood of uninjured mice demonstrated a progressive reduction in tdTom+ leukocytes (C) or monocytes (D). (E) Flow quantification of the blood of injured mice demonstrated no difference in reporter expression in CD45+ leukocytes. n = 4 (B), n = 3 to 4 (C to E). Error bars indicate ± SEM. DPI, days post-LPC injection. Scale bars, 25 μm.

  • Fig. 2 Single-cell transcriptome-wide profiling of adult microglia/CAMs.

    (A) FACS purification and single-cell sequencing of microglia/CAMs from adult CX3CR1creER; Rosa26tdTom mice. (B) Unsupervised graph-based clustering of single-cell RNA-seq dataset projected onto a tSNE plot. Most tdTom+ microglia/CAMs from lesioned sample aggregated distinctly away from tdTom+ microglia/CAMs from naïve sample (B, box) and formed distinct clusters (lesions 1, 2, and 3) compared to naïve (naïve 1 and 2) (C). (D) Heat map of top 10 marker genes (determined by likelihood-ratio test) for each of the five clusters. (E) Pseudotime two-dimensional minimal spanning tree clustering to understand potential intermediary microglial/CAMs states. Each dot represents single cells ordered in pseudotime color-coded either by sample (box) or by Seurat-state (naïve 1 or 2, lesion 1, 2, or 3) or pseudotime state (blue). The line connecting the dots outlines a path of transcriptional relatedness, which represents fate trajectory. Uninjured naïve branch was set as the root state to specify the start point of the trajectory (dark blue). There was a bifurcation into two subpopulations (cell fates 1 and 2) emanating after injury (light blue). (F) Top 50 pseudotime-dependent genes calculated from the full trajectory seen in (E) (q value, <1 × 10−27). Legend represents arbitrary units based on the order of single cells (the genes representing cells in the most extreme states are darker in color and are assigned a value of ±3).

  • Fig. 3 Single-cell GRN profiling of adult microglia/resident macrophage and activated macrophages.

    (A) Unsupervised graph-based clustering of single-cell RNA-seq dataset projected onto a tSNE plot comparing microglia isolated from naïve and LPC-lesioned mice (B) GRN density projected onto the tSNE plot. (C) Raw gene expression (left), MAGIC-imputed gene expression (center), and SCENIC analysis (right) demonstrated that injury-associated microglia/resident macrophages were distinct from uninjured conditions. (D) It was revealed that there were 245 regulons, of which several were unique, largely to the uninjured state.

  • Fig. 4 Microglia progressively outnumber macrophages after CNS demyelination.

    (A) Schematic depicting experimental plan. (B to G) Representative immunohistochemical images of the lesioned spinal cord (CNS; B to D) and sciatic nerve (PNS; E to G) demonstrating widespread reporter expression (red) in the CNS versus the PNS. CD45 (green); 4′,6-diamidino-2-phenylindole (DAPI) (blue). This observation was also reflected in quantification (CNS: H and J; PNS: I and K). n = 4 (0 DPI) or n = 5 to 9 (H to K); analysis of variance (ANOVA) with Tukey’s multiple comparison test (P < 0.05; H and I), two-way ANOVA with Sidak’s multiple comparison test (P < 0.05; J and K). Error bars indicate ± SEM. Scale bars, 20 μm.

  • Fig. 5 Preferential microglia proliferation and infiltrating macrophage apoptosis in the lesioned CNS.

    (A and C) Representative immunohistochemical images of the lesioned spinal cord (A) and sciatic nerve (C) demonstrating more proliferative (Ki67+, green) reporter-positive cells (yellow, arrowheads) compared to reporter-negative cells (white, boxes) in the CNS and no difference between cell types in the PNS. This observation was also reflected in quantification (*P < 0.02) (B and D). (E and F) Representative immunohistochemical images of the lesioned spinal cord (E) and sciatic nerve (F) at 3 days after LPC demonstrating more apoptotic (CC3+ green) infiltrating macrophage (CD45+ tdTom, yellow, arrowheads) in the CNS versus the PNS. This observation was also reflected in quantification (*P < 0.03; G and H). (I to K) Using conditioned media (CM) from spinal cord (CNS) and sciatic nerve (PNS) on BMDM, we demonstrated a reduction in cell numbers (J) and an increase in apoptosis (CC3+ cells; K) in CNS-primed media conditions compared to PNS-primed media. n = 6 to 7, *P < 0.02. (B), n = 3 to 4 (D); two-way ANOVA with Sidak’s multiple comparison test; n = 4 (PNS) and 9 (CNS) two-way ANOVA with Sidak’s multiple comparison test (G and H); n = 6 (two independent experiments) ANOVA with Tukey’s multiple comparison test (I to K); Error bars indicate ± SEM. Scale bar, 20 μm. FOV, field of view.

  • Fig. 6 Microglia limit toxicity and expansion into the spared white matter of CNS-infiltrating macrophages following LPC demyelination.

    Representative immunohistochemical images of the lesioned spinal cord at 3 and 7 days after LPC (A) demonstrating confinement of monocyte-derived cells (CD45+ tdTom, white) by microglia (red) apparent by 7 days after LPC (high resolution) (B). This observation was also reflected in quantification as the proportion of lesions with the confinement phenotype (C). Representative immunohistochemical images of the lesioned spinal cord in nonablated iCX3CR1tdTom;WT (D) and DT-ablated iCX3CR1tdTom;iDTR (E) mice with quantification of microglia ablation showing no significant compensation from infiltrating macrophages (F and G). Microglia/CAM ablation demonstrated an increase in CNS-infiltrating macrophages (CD45+ tdTom) into the spared white matter (H) and a reduction in lesional axons (I). (J) Schematic depicting RNA-seq experimental plan. RNA-seq analysis of laser-captured microdissected lesions revealed that 10 of 13 infiltrating macrophage-associated genes were elevated after microglia ablation in the LPC lesion (K). In contrast, microglia-specific genes were reduced on average of 70% following microglia ablation (L). n = 8 to 9 (C), χ2 test; n = 7 to 8 (J), n = 3 to 4 samples each with two to five pooled animals, Mann-Whitney test; error bars indicate ± SEM. White dashed line is lesion edge. Scale bar, 20 μm. *P < 0.05.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/3/eaay6324/DC1

    Fig. S1. Minimal expression of Cx3Cr1creER-TdTom in monocytes or macrophages in the spleen and lymph node.

    Fig. S2. Lack of meningeal or perivascular macrophage marker Lyve1.

    Fig. S3. Common markers to distinguish microglia and macrophages are less sensitive after microglia activation.

    Fig. S4. Gating strategy for flow cytometry.

    Fig. S5. Microglia express CX3CR1 and tdTomato.

    Fig. S6. RNA-seq experiment metrics and quality control.

    Fig. S7. Sparse CAM present following single-cell RNA sequencing of fate mapped cells (tdTom+).

    Fig. S8. Activated microglia express ApoE.

    Fig. S9. M1 and M2 genes in scRNA sequencing dataset.

    Fig. S10. Distinct activated microglia subphenotypes.

    Fig. S11. Fate mapping as a tool to specifically label resident macrophage in sciatic nerve.

    Fig. S12. CNS and PNS LPC injections.

    Fig. S13. Infiltrating macrophages expand in CNS when microglia/CAMs are ablated following LPC demyelination.

    Fig. S14. Cytosolic pattern recognition receptors reduced in the absence of microglia.

    Fig. S15. IFN type I and type II reduced in the absence of microglia.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Minimal expression of Cx3Cr1creER-TdTom in monocytes or macrophages in the spleen and lymph node.
    • Fig. S2. Lack of meningeal or perivascular macrophage marker Lyve1.
    • Fig. S3. Common markers to distinguish microglia and macrophages are less sensitive after microglia activation.
    • Fig. S4. Gating strategy for flow cytometry.
    • Fig. S5. Microglia express CX3CR1 and tdTomato.
    • Fig. S6. RNA-seq experiment metrics and quality control.
    • Fig. S7. Sparse CAM present following single-cell RNA sequencing of fate mapped cells (tdTom+).
    • Fig. S8. Activated microglia express ApoE.
    • Fig. S9. M1 and M2 genes in scRNA sequencing dataset.
    • Fig. S10. Distinct activated microglia subphenotypes.
    • Fig. S11. Fate mapping as a tool to specifically label resident macrophage in sciatic nerve.
    • Fig. S12. CNS and PNS LPC injections.
    • Fig. S13. Infiltrating macrophages expand in CNS when microglia/CAMs are ablated following LPC demyelination.
    • Fig. S14. Cytosolic pattern recognition receptors reduced in the absence of microglia.
    • Fig. S15. IFN type I and type II reduced in the absence of microglia.

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