M2 microglia and macrophages drive oligodendrocyte differentiation during CNS remyelination

Abstract

The lack of therapies for progressive multiple sclerosis highlights the need to understand the regenerative process of remyelination that can follow CNS demyelination. This involves an innate immune response consisting of microglia and macrophages, which can be polarized to distinct functional phenotypes: pro-inflammatory (M1) and anti-inflammatory or immunoregulatory (M2). We found that a switch from an M1- to an M2-dominant response occurred in microglia and peripherally derived macrophages as remyelination started. Oligodendrocyte differentiation was enhanced in vitro with M2 cell conditioned media and impaired in vivo following intra-lesional M2 cell depletion. M2 cell densities were increased in lesions of aged mice in which remyelination was enhanced by parabiotic coupling to a younger mouse and in multiple sclerosis lesions that normally show remyelination. Blocking M2 cell-derived activin-A inhibited oligodendrocyte differentiation during remyelination in cerebellar slice cultures. Thus, our results indicate that M2 cell polarization is essential for efficient remyelination and identify activin-A as a therapeutic target for CNS regeneration.

Figure 1. A switch from an M1 to an M2-dominant microglia/macrophage response occurs at the initiation of remyelination.

(a) Oligodendroglial lineage cell responses in the corpus callosum at 3, 10, and 21 days post lesion (dpl) induction by stereotactic injection of lysolecithin (LPC). (b) Lesions immunostained against iNOS (green), Arg-1 (red), and CD68 (white) at 3, 10 and 21 dpl. Inset: isotype controls. Scale bar, 25 μm. (c) Percentage of iNOS+ M1 cells, Arg-1+ M2 cells, and iNOS-Arg-1-(unpolarized) cells per field in lesion ± s.e.m.. One way ANOVA and Newman-Keuls post-hoc test, ***p<0.001 (n=5 mice, df=44).

Figure 2. Microglia and peripherally-derived macrophages contribute to both M1 and M2 polarized populations during remyelination.

(a) Ccr2^−/− mice were lesioned in the corpus callosum to examine polarization in microglia only. (b) Ccr2^−/− lesions at 3 and 10 dpl immunostained against iNOS (green), Arg-1 (red) and CD68 (white). Scale bar, 25 μm. (c) Percentage of iNOS+, Arg-1+, or unpolarized (iNOS-, Arg-1-) cells in Ccr2^−/− lesions ± s.e.m.. One way ANOVA and Newman-Keuls post-hoc test, ***p<0.001 (n=2-3 mice, df=14). (d) Ccr2^−/−mice were lesioned in the corpus callosum and injected with GFP-expressing wildtype bone marrow derived cells to examine polarization in peripherally-derived macrophages only. (e) GFP+ macrophages (stars) and GFP-microglia (hash symbols) were iNOS+ (white) or Arg-1+ (red). Scale bar, 5 μm. (f) Numbers of GFP+ (macrophages) (black bars) and GFP-(microglia)(white bars) iNOS+ M1 and Arg-1+ M2 cells per field ± s.e.m. (n=3 mice).

Figure 3. M2 conditioned media promotes oligodendrocyte differentiation.

(a) OPCs treated with unconditioned (control) or M1, M2a, or M2c conditioned media immunostained against NG2 (green) and MBP (red). Scale bar, 50 μm. (b) Percentage of MBP+ cells ± s.e.m. following treatment with microglia conditioned media, or polarizing factors alone as a control., Kruskal-Wallis test and Dunn’s multiple comparison post-test, ***p<0.001 (n=3 separate experiments). (c) Number of MOG+ cells per field ± s.e.m. following treatment with microglia conditioned media. Kruskal-Wallis test and Dunn’s multiple comparison post-test, **p<0.01 (n=3 separate experiments).

Figure 4. Selective depletion of M1 microglia/macrophages in a demyelinated lesion in the CNS impairs OPC proliferation.

(a) Gadolinium chloride (GdCl3) was injected into corpus callosum lesions at the onset of demyelination (0 dpl) prior to the peak in M1 polarization at 3 dpl. (b) Representative images of control or GdCl3-injected lesions at 3 dpl with immunostaining against iNOS (green), Arg-1 (red), and CD68 (white). Scale bar, 25 μm. (c) Numbers of iNOS+ M1, Arg-1+ M2, or unpolarized (iNOS-, Arg-1-) cells per field ± s.e.m. in control and GdCl3-injected lesions at 3 dpl. One way ANOVA and Newman-Keuls post-test, **p<0.01 (n=5 mice, df=29). (d) Density of PCNA+ Nkx2.2+ proliferating OPCs per mm² in control and GdCl3-injected lesions ± s.e.m. at 3 dpl. P=0.0496, 2-tailed Student’s t-test (n=5 mice, df=8).

Figure 5. Selective depletion of M2 microglia/macrophages in a demyelinated lesion in the CNS impairs oligodendrocyte differentiation.

(a) Mannosylated clodronate liposomes were used to induce apoptosis selectively in M2 polarized microglia/macrophages due to upregulation of mannose receptor, and induce clodronate-mediated apoptosis following phagocytosis. (b) Corpus callosum lesions were injected with MCLs at 8 dpl prior to the peak in M2 polarization at 10 dpl. (c) Representative images at 10 dpl of control and MCL-injected lesions immunostained for iNOS (green), Arg-1 (red), and CD68 (white). Scale bar, 25 μm. (d) Number of iNOS+ M1, Arg-1+ M2, or unpolarized (iNOS-, Arg-1-) cells per field ± s.e.m. in control and MCL-injected lesions at 10 dpl. One way ANOVA and Newman-Keuls post-test, **p<0.01, ***p<0.001 (n=5 mice, df=29). (e) Mean lesion pixel counts ± s.e.m. for MAG and MBP at 10 dpl. 2-tailed Student’s t-test versus control, P=0.0361 and 0.0106, respectively (n=4-5 mice, df=7). (f) Representative images of lesions (dotted outline) at 10 dpl in control and MCL-injected lesions immunostained against MBP (red) and MAG (green). Scale bar, 100 μm. (g) Quantification of nodes of Ranvier (paranodal Caspr expression flanking nodal Ankyrin-G expression) ± s.e.m. in control and MCL-treated lesions at 21 dpl. 2-tailed Student’s t-test versus control, P=0.0068 (n=6 mice, df=10). Box plots indicate median (centre line), 25^th -75^th percentile (box limits) and minimum and maximum (whiskers). (h) Representative images of control and MCL-treated lesions at 21 dpl immunostained against Caspr (green) and Ankyrin-G (red). Scale bar, 25 μm.

Figure 6. Restored remyelination efficiency in aged mice via heterochronic parabiosis is associated with increased densities of M2 polarized cells.

(a) Lesion induction in the ventral spinal cord by lysolecithin injection. Parabiotic pairings between young (Y-Y) (white) or old (O-O) (dark gray) animals were compared to lesions in old animals following heterochronic pairing (Y-O) (light gray), with previous studies showing restored remyelination efficiency in old partners of Y-O pairings. (b) Lesions immunostained at 7 dpl against Arg-1 (red) and CD68 (white). Scale bar, 25 μm. (c) MR+ IB4+ M2 cells/0.1 mm² ± s.e.m. at 7 dpl. 2-tailed Student’s t-test: Y/Y vs. O/O, P=0.048; Y/O vs. O/O, P=0.005 (n=4 (O/O), 6(Y/O), 4 (Y/Y), df=13). (d) CD16/32+ IB4+ cells/0.1 mm² ± s.e.m. at 7 dpl. 2-tailed Student’s t-test: Y/Y vs. O/O, P=0.0027; Y/Y vs. Y/O, P=0.014 (n=4 (O/O), 6(Y/O), 4 (Y/Y), df=13). (e) Parabiosis between a GFP-expressing young mouse and wildtype (WT) old mouse with a spinal cord lesion. (f) GFP expressing iNOS+ CD68+ M1 macrophages and Arg-1+ CD68+ M2 macrophages. Scale bar, 5 μm.

Figure 7. M2 microglia/macrophage densities are increased in acute active and the rim of chronic active multiple sclerosis lesions.

(a) Total CD68+ microglia/macrophages/mm² were significantly increased in acute active (P=0.001), chronic active rim (P=0.0156) and centre (P=0.0156), chronic inactive (P=0.001) and remyelinated lesions (P=0.002). (b) iNOS+ M1 microglia/macrophages/mm² were increased in acute active (P=0.001), chronic active rim (P=0.0156) and centre (P=0.0313), chronic inactive (P=0.0005), and remyelinated lesions (P<0.0001). (c) MR+ M2 microglia/macrophages/mm² were increased in acute active lesions (P=0.0068) and the rim of chronic active lesions (P=0.0156). Mann-Whitney test. n for each lesion type indicated in Supplementary Table 1 online. (d) Representative image of MS lesion with iNOS immunolabeling. Scale bar, 100 μm. (e) Representative image of MS lesion with MR immunolabeling. Scale bar, 100 μm. (f) Co-localization of MR (top) and iNOS (bottom) with microglia/macrophage marker CD68 (arrowheads). Scale bar, 25 μm. (g) MR+ CD68+ M2 cells (arrows) were sometimes associated with blood vessels. Scale bar, 25 μm.

Figure 8. Activin-A is an M2-derived factor that drives oligodendrocyte differentiation during remyelination.

(a) Percentage of activin-A immunopositive cells per field in M1, M2a, and M2c microglia ± s.e.m. (b) Control corpus callosum lesions at 3 and 10 dpl (top and middle panels) and MCL-injected lesion at 10 dpl (bottom panel) immunostained for activin-A (red) and iNOS or MR (green). Scale bar, 25 μm. (c) Representative images of NG2+ OPCs (green) in remyelinating lesions expressing activin receptors Acvr2A, Acvr2B, and Acvr1B (red; arrows). Scale bar, 10 μm. (d) Percentage of MBP+ cells ± s.e.m. in OPC cultures treated with activin-A. One way ANOVA and Newman-Keuls post-hoc test, *p<0.05 (n=4 separate experiments, df=15). (e) Percentage of MBP+ cells ± s.e.m. in OPC cultures treated with combinations of M2a conditioned media, goat isotype control IgG, or goat anti-activin-A antibody. One way ANOVA and Newman-Keuls post-hoc test, **p<0.01 (n=4 separate experiments, df=11), ns=p>0.05. (f) 300 μm sagittal sections of cerebellum and attached hindbrain were taken from newborn mouse brain to obtain organotypic cultures, demyelinated with lysolecithin (LPC) and treated with M2 conditioned media and either anti-activin-A IgG or control goat IgG. (g) Representative images of slices treated with M2 conditioned media and goat IgG (left) or anti-activin-A antibody (right) immunostained for CC1 (white) and MBP (red). Scale bar, 25 μm. (h) Percentage of CC1+ oligodendrocytes normalized to values obtained from slices treated with IgG alone. 2-tailed one-sample or Student’s t-test (respectively), M2 conditioned media + IgG vs. IgG, P=0.0213; M2 conditioned media + IgG vs. M2 conditioned media + anti-activin-A IgG, P=0.0071 (n=8 slices, df=14). Box plots indicate median (centre line), 25^th-75^th percentile (box limits), minimum and maximum (whiskers).